Bi-directional sequencing compositions and methods

ABSTRACT

In some embodiments, methods for obtaining sequence information from a nucleic acid template linked to a support include hybridizing a first primer to a template strand linked to a support, sequencing a portion of the nucleic acid template, thereby forming an extended first primer product that is complementary to a portion of the nucleic acid template, In some embodiments, the method further includes introducing a nick into a portion of the template strand that is hybridized to the extended first primer product, degrading a portion of the template strand from the nick using a degrading agent, where a portion of the extended first primer remains hybridized to an undegraded portion of the template strand, and sequencing at least some of the single-stranded portion of the extended first primer by synthesis.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of application Ser. No. 13/543,521,filed Jul. 6, 2012, now U.S. Pat. No. 9,139,874, which claims benefit ofpriority under 35 U.S.C. §119(e) to U.S. Provisional Application No.61/505,420, filed Jul. 7, 2011, U.S. Provisional Application No.61/544,992, filed Oct. 7, 2011, U.S. Provisional Application No.61/562,252, filed Nov. 21, 2011, and U.S. Provisional Application No.61/577,637 filed Dec. 19, 2011 entitled “SEQUENCING METHOD ANDCOMPOSITIONS”, the disclosures of which are incorporated herein byreference in their entireties.

SEQUENCE LISTING

This application hereby incorporates by reference the material of theelectronic Sequence Listing filed concurrently herewith. The material inthe electronic Sequence listing is submitted as a text (.txt) fileentitled “LT00545_ST25.txt” created on Jul. 3, 2012, which has a filesize of 3 KB, and is herein incorporated by reference in its entirety.

Throughout this application various publications, patents, and/or patentapplications are referenced. The disclosures of these publications,patents, and/or patent applications in their entireties are herebyincorporated by reference into this application in order to more fullydescribe the state of the art to which this invention pertains.

FIELD

In some embodiments, the disclosure relates generally to methods,systems, compositions and kits useful for obtaining sequence informationfrom a nucleic acid molecule. In some embodiments, the methods, systems,compositions and kits are useful for bi-directional sequencing ofnucleic acids. In some embodiments, the methods, systems, compositionsand kits are useful for generating single stranded polynucleotides. Thedisclosure also generally relates to sequencing nucleic acids in variousorientations, including orientations that are reversed relative to eachother. The methods can include sequencing-by-synthesis, paired-endsequencing, or both.

INTRODUCTION

Many nucleic acid sequencing methods involve sequencing-by-synthesis,wherein nucleic acid synthesis is performed via serial incorporation ofnucleotides in a template-dependent fashion, typically using apolymerase, and the identity and order of incorporated nucleotides isdetermined. Recently, label-free methods of sequencing-by-synthesis havebeen developed, including so-called “ion based” sequencing, wherein oneor more byproducts of nucleotide incorporation can be detectedelectronically using chemically sensitive transistors (e.g., FETsincluding chemFETs or ISFETs). When performing nucleic acid sequencing,it is frequently desirable to sequence the template strand in bothdirections. Such bi-directional sequencing can be helpful in increasingthe total amount of sequence information available from a given templatestrand, especially when the length of the template strand exceeds theread lengths typically obtainable from the sequencing method employed.Even in cases where the read lengths of the sequencing method are of thesame order as template length, bi-directional sequencing can provideindependent validation of sequence information by allowing comparison ofthe “forward” with the corresponding “reverse” sequence obtained bysequencing the template strand (or its complement) in the oppositedirection. While various methods of bi-directional or “paired end”sequencing are known, there remains a need for improved methods,compositions, systems, apparatuses and kits that allow bi-directionalsequencing of a template strand.

SUMMARY

Provided herein are compositions, systems, methods and kits forobtaining sequence information from one or more nucleic acid molecules,for example via nucleic acid sequencing.

In some embodiments, the sequencing includes bi-directional sequencingof a nucleic acid molecule of interest.

In some embodiments, the sequencing includes sequencing and obtainingsequencing information in a forward direction, followed by sequencingand obtaining sequence information in a direction that is reversedrelative to the forward direction.

In some embodiments, bi-directional sequencing of a nucleic acidmolecule can be performed at a single location or at multiple locationsalong a nucleic acid strand.

In some embodiments, the disclosure relates generally to methods,compositions, systems, apparatuses and kits for obtaining sequenceinformation from a nucleic acid. In some embodiments, the methodincludes obtaining sequence information from a portion of a nucleicacid. In some embodiments, the method can include obtaining sequencinginformation for substantially the entire length of the nucleic acid. Insome embodiments, obtaining sequencing information can includedetermining the nucleotide identity of one or more nucleotides along thelength of the nucleic acid. In some embodiments, obtaining sequencinginformation can include determining the nucleotide identity ofsubstantially all of the nucleotides along the length of the nucleicacid. In some embodiments, obtaining sequencing information can includedetermining the nucleotide identity of substantially all of thenucleotides along the length of the nucleic acid in a first orientationand a second (reversed) orientation relative to the first orientation.In some embodiments, obtaining sequencing information can includedetermining the nucleotide identity of one or more nucleotides along thelength of the nucleic acid in a paired-end sequencing orientation. Insome embodiments, obtaining sequencing information can includedetermining the nucleotide identity of one or more nucleotides along thelength of the nucleic acid in a bi-directional sequencing orientation.

In some embodiments, the disclosure relates generally to methods,compositions, systems, apparatuses and kits for obtaining sequenceinformation from at least a portion of a nucleic acid. In someembodiments, obtaining sequencing information can include sequencing bylabel-free or ion based sequencing methods. In some embodiments,obtaining sequencing information can include labeled or opticallydetectable based sequencing methods such a fluorescence orbioluminescence. In some embodiments, obtaining sequencing informationcan include determining the identity of an incorporated nucleotide bymonitoring sequencing reaction byproducts released during nucleotideincorporation. In some embodiments, the sequencing reaction byproductsreleased during nucleotide incorporation can include hydrogen ions,inorganic pyrophosphate or inorganic phosphate.

In some embodiments, the disclosure relates generally to methods,compositions, systems, apparatuses and kits for obtaining sequenceinformation from a nucleic acid via paired-end sequencing. In someembodiments, the nucleic acid can include a DNA, RNA, cDNA, mRNA, orDNA/RNA hybrid. In some embodiments, the nucleic acid can be atarget-specific nucleic acid associated with genotyping, such as anucleic acid containing a single nucleotide polymorphism or a shorttandem repeat. In some embodiments, the nucleic acid can be atarget-specific nucleic acid associated with one or more medicallyrelevant or medically actionable mutations, such as mutations associatedwith cancer or inherited disease. In some embodiments, the nucleic acidcan be derived from a mammal such as a human.

In some embodiments, the method (and related compositions, systems,apparatuses and kits using the disclosed methods) can include obtainingsequencing information from a nucleic acid linked to a support.Optionally, the support can include any suitable support such as, butnot limited to a bead, particle, microparticle, microsphere, slide,flowcell or reaction chamber. In some embodiments, the support caninclude a solid support. In some embodiments, the support can include aplanar support such as a flowcell or slide. In some embodiments, thesupport can include an Ion Sphere Particle (ISP). In some embodiments,the nucleic acid includes a template strand. In some embodiments, thetemplate strand can further include one or more adaptors. In someembodiments, the one or more adaptors can optionally include a barcodeor tagging sequence. In some embodiments, a template strand including anadaptor can further include one or more nucleotide residues that areresistant to a degrading agent. In some embodiments, an adaptor caninclude one or more phosphorothioate or 2-O-Methyl RNA (2′ OMe)nucleotides. In some embodiments, the template strand can be linked to asupport through the 5′ end of the template strand. In some embodiments,the template strand can be linked to the support through at least onenucleotide in the template strand that is situated 5′ of a nick site inthe template strand. In some embodiments, the at least one nucleotide inthe template strand situated 5′ of the nick site can be resistant to adegrading agent. In some embodiments, the at least one nucleotideresistant to a degrading agent includes a phosphorothioate, a 2′OMeresidue, or a combination thereof. In some embodiments, the at least onenucleotide resistant to a degrading agent can be located at the 5′ endof the template strand, which can optionally include an adaptor. In someembodiments, an adaptor can include the at least one nucleotideresistant to a degrading agent. In some embodiments, an adaptor can linkthe template strand to the support.

In some embodiments, the disclosure relates generally to methods,compositions, systems, apparatuses and kits for obtaining sequenceinformation from a nucleic acid comprising hybridizing a first primer tothe nucleic acid linked to a support and sequencing a portion of thenucleic acid. In some embodiments, the method includes a first primerthat is complementary or substantially complementary (e.g.,target-specific) to the nucleic acid linked to the support. In someembodiments, the first primer can include a universal or shared primersequence. In some embodiments, the first primer can include a degenerateprimer sequence. In some embodiments, the first primer includes one ormore nucleotide analogs. In some embodiments, the first primer ishybridized to the nucleic acid under stringent hybridization conditions.In some embodiments, the first primer contains one or more residues ornucleotides that are resistant to degradation by a degrading agent. Insome embodiments, the extended first primer product contains one or moreresidues or nucleotides that are resistant to degradation by a degradingagent. In some embodiments, the extended first primer product or firstprimer is nuclease resistant. In some embodiments, the first primercontains one or more residues or nucleotides that are resistant todegradation by an exonuclease. In some embodiments, the first primercontains one or more phosphorothioates, 2′OMe residues, or a combinationthereof.

In some embodiments, the first primer is hybridized to the distal end ofa nucleic acid, where the proximal end of the nucleic acid is attachedto a support. In some embodiments, the first primer is hybridized to theproximal end of a nucleic acid, where the distal end of the nucleic acidis attached to a support. In some embodiments, the first primer isextended via template-dependent nucleic acid synthesis. In someembodiments, the method includes sequencing by synthesis. In someembodiments, the method includes sequencing by ligation. In someembodiments, the method includes sequencing by hybridization. In someembodiments, the sequencing includes sequencing via template dependentnucleic acid synthesis.

In some embodiments, the method includes forming an extended firstprimer product that is complementary or substantially complementary to aportion of the nucleic acid. In some embodiments, the extended firstprimer product is complementary or substantially complementary to all ofthe nucleotides in the nucleic acid. In some embodiments, the sequencinginformation obtained can determine substantially all of the nucleotidesin the nucleic acid or the extended first primer product. In someembodiments, the extended first primer product can be cross-linked to asupport. In some embodiments, the extended first primer product can becross-linked to a support that is different to a support linked to thetemplate strand. In some embodiments, the extended primer product can beseparated from the template strand prior to cross-linking to a support.In some embodiments, the extended primer product can be cross-linked toa support prior to introducing the nick or degrading step. In someembodiments, the extended primer product can be cross-linked to asupport shared with the template strand. In some embodiments, theextended primer product can be photo-chemically cross-linked to asupport. In some embodiments, the template strand can be removed ordegraded after linking the extended primer product to a support.

In some embodiments, the method can include sequencing a portion anucleic acid template by synthesis, where the sequencing by synthesisincludes extending the first primer via template-dependent nucleic acidsynthesis. In some embodiments, the method can include sequencing aportion of a nucleic acid, optionally linked to a support, where thesequencing includes extending the first primer via sequencing byhybridization or sequencing by ligation. In some embodiments, thesequencing can include sequencing at least some of the single-strandedportion of the extended first primer by extending the free 3′ end of thenick. In some embodiments, extending the free 3′ end of the nick caninclude sequencing via nucleic acid synthesis, thereby synthesizing anucleic acid molecule that is complementary to at least some of thesingle-stranded portion of the extended first primer. In someembodiments, sequencing at least some of the single-stranded portion ofthe extended first primer product can include hybridizing a reverseprimer to a sequence within the single-stranded portion of the extendedfirst primer product. In some embodiments, hybridizing a reverse primerto a sequence within the single stranded portion of the extended firstprimer product can include extending the reverse primer using apolymerase. In some embodiments, the reverse primer can include one ormore nucleotides resistant to degradation by a degrading agent. In someembodiments, the reverse primer is nuclease resistant. In someembodiments, the reverse primer contains one or more residues ornucleotides that are resistant to degradation by an exonuclease. In someembodiments, the reverse primer contains one or more phosphorothioates,2′OMe residues, or a combination thereof.

In some embodiments, the method (and related kits, compositions andapparatus using the disclosed methods) can include introducing a nickinto a portion of the template strand that is hybridized to the extendedfirst primer product. In some embodiments, the nick includes a free 5′end and a free 3′ end in the template strand. In some embodiments, aplurality of nicks can be introduced into the portion of the templatestrand that is hybridized to the extended first primer. In someembodiments, introducing one or more nicks can include introducing a gapalong the template strand. In some embodiments, introducing asite-specific nick can include introducing a site-specific gap along thetemplate strand. In some embodiments, one or more nicks can beintroduced randomly along the length of the template strand. In someembodiments, one or more nicks can be introduced at site-specificlocations along the length of the template strand. In some embodiments,one or more nicks can be introduced into the extended primer product andthe template strand. In some embodiments, one or more nicks can beintroduced into the template strand, which optionally includes one ormore adaptors. In some embodiments, one or more nicks can be introducedinto the template strand and the extended primer product, however it ispreferred that the nicks are not positioned as to introduce a doublestranded break. In some embodiments, a nickase can be used to introduceone or more nicks in the template strand or the extended primer product.In some embodiments, a restriction enzyme can be used to introduce oneor more nicks into the template strand or extended primer product. Insome embodiments, one or more nicks can be introduced into the templatestrand using a uracil DNA glycosylase. In some embodiments, the templatestrand can include at least one adaptor, where the adaptor can includeone or more nicks.

In some embodiments, the method can further include degrading a portionof the template strand from the free 5′ end of the nick using adegrading agent. In some embodiments, the method can include degrading aportion of the template strand from the free 5′ end of the nick using adegrading agent, thereby generating a single-stranded portion of theextended first primer product. In some embodiments, the degrading agentcan include an enzymatic, thermal or chemical treatment. In someembodiments, the degrading agent can include an exonuclease. Optionally,the degrading agent can include a 5′-3′ exonuclease. In someembodiments, the degrading can further include digesting the templatestrand from the free 5′ end of the nick using the 5′-3′ exonuclease. Insome embodiments, the degrading agent can include an exonuclease incombination with an endonuclease. In some embodiments, the degradingagent can include a heat treatment.

In some embodiments, a portion of the extended first primer product canremain hybridized to an undegraded portion of the template strand. Insome embodiments, the extended first primer product that remainshybridized to an undegraded portion of the template strand can undergosequencing. In some embodiments, the extended first primer product thatremains hybridized to an undegraded portion of the template can undergoseparation from the undegraded template prior to sequencing. In someembodiments, a separated extended first primer product can be capturedby a binding partner or capture probe. In some embodiments, a capturedor bound extended primer product can be sequenced by any suitablesequencing means. In some embodiments, the sequencing can includesequencing by synthesis, sequencing by ligation and/or sequencing byhybridization.

In some embodiments, the disclosure relates generally to methods,compositions, systems, apparatuses and kits for nucleic acid sequencing.In some embodiments, the method (and related kits, compositions andapparatuses using the method) can include hybridizing a first primer toa distal end of a nucleic acid strand having a distal and proximal end,where the proximal end of the nucleic acid strand is linked to a solidsupport. In some embodiments, the method can further include extendingthe hybridized first primer in the direction of the proximal end of thenucleic acid strand and the solid support, thereby forming an extendedfirst primer product that is complementary to a portion of the nucleicacid strand and obtaining a first sequencing read. In some embodiments,the method can further include introducing a site-specific nick into theproximal end of the nucleic strand hybridized to the extended firstprimer product. Optionally, the method can further include degrading aportion of the nucleic acid strand, thereby generating a single-strandedportion within the extended first primer product, wherein a portion ofthe extended first primer product remains hybridized to the nucleic acidstrand. In some embodiments, the method can further include extendingthe single-stranded portion within the extended first primer product,thereby obtaining a second sequencing read. In some embodiments, themethod can further include consolidating the first and second sequencingread. In some embodiments, the consolation can including aligning thefirst and second sequencing read against a reference sample. In someembodiments, the reference sample is a human sample. In someembodiments, the reference sample is hg19. In some embodiments, thealigning can identity a mismatch, deletion, insertion or translocationor variations in the nucleic acid strand. In some embodiments, aligningthe first and second sequencing reads against a reference sequence candetermine the presence of a deletion, insertion, translocation,inversion, variation, mutation or mismatch in the nucleic acid strand.

In some embodiments, the disclosure relates generally to methods,compositions, systems and apparatuses for bi-directional sequencing of anucleic acid. In some embodiments, a method for obtaining bi-directionalsequence information from a nucleic acid molecule can includehybridizing a first primer to a nucleic acid molecule, sequencing aportion of the nucleic acid molecule, wherein the sequencing includesextending the first primer, thereby forming an extended first primerproduct that is complementary to a portion of the nucleic acid molecule.In some embodiments, the extended first primer product can besubstantially complementary to the nucleic acid molecule. In someembodiments, the extending can include extending via template-dependentnucleic acid synthesis. In some embodiments, the method can furtherinclude introducing a nick into a portion of the nucleic acid moleculethat is hybridized to the extended first primer product. In someembodiments, a plurality of nicks can be introduced into the nucleicacid molecule. In some embodiments, one or more nicks are introducedinto the 5′end of the nucleic acid molecule. In some embodiments, thenick can include a free 5′ end and a free 3′ end in the nucleic acidmolecule. In some embodiments, the method can further include degradinga portion of the nucleic acid molecule from the free 5′ end of the nickusing a degrading agent, thereby generating a single-stranded portion ofthe extended first primer product. In some embodiments, the extendedfirst primer product can remain hybridized to an undegraded portion ofthe nucleic acid molecule. In some embodiments, the method furtherincludes sequencing at least some of the single-stranded portion of theextended first primer product. In some embodiments, the sequencing caninclude sequencing by synthesis, sequencing by ligation and/orsequencing by ligation. In some embodiments, the nucleic acid moleculesis linked to a support, such as but not limited to a solid support. Insome embodiments, the nucleic acid molecule is a single-strandedtemplate. In some embodiments, the nucleic acid molecule is adouble-stranded template that can undergo a denaturation treatment toform a single stranded template.

In some embodiments, the disclosure relates generally to kits (andmethods, compositions, apparatuses and systems that use the kits)including a primer having an exonuclease resistant nucleotide sequencesubstantially complementary to a template nucleic acid to be sequenced.In some embodiments, the kit can include a primer that is complementaryto the template nucleic acid to be sequenced. In some embodiments, theprimer can include one or more phosphorothioate or 2′ OMe residues. Insome embodiments, the kit can further include one or more polymerases,one or more dNTPs, one or more nicking enzymes and one or more degradingenzymes. In some embodiments, the kit can include one or more DNApolymerases, such as a native polymerase, mutant polymerase, geneticallyengineered DNA polymerase or fragment thereof, where the fragment iscapable of catalyzing polymerization. In some embodiments, thepolymerase can include a thermostable polymerase. In some embodiments,the kit can include one or more ddNTPs for terminating polymerization.In some embodiments, the nicking agent can include a nickase. In someembodiments, the nicking agent can be a uracil DNA glycosylase. In someembodiments, the degrading agent can include an exonuclease, optionallyin combination with an endonuclease. In some embodiments, the kit canfurther include one or more buffers, cations, salts, additives, reducingagents and/or supports. In some embodiments, the kit can further includea capture or binding partner. In some embodiments, the kit can furtherinclude a standard or control nucleic acid molecule and/or instructionsas to use the kit.

In some embodiments, the disclosure relates generally to methods (andrelated compositions, kits, systems and apparatuses using the methods)for improving nucleic acid sequencing accuracy. In some embodiments, themethod includes hybridizing a first primer to a distal end of a nucleicacid strand having a distal and proximal end. Optionally, the methodincludes linking the proximal end of the nucleic acid strand to a solidsupport. In some embodiments, the method further includes extending thehybridized first primer in the direction of the proximal end of thenucleic acid strand (and optionally the solid support), thereby formingan extended first primer product that is complementary to a portion ofthe nucleic acid strand and obtaining a first sequencing read. In someembodiments, the method can further include introducing a site-specificnick into the proximal end of the nucleic strand hybridized to theextended first primer product. In some embodiments, a site-specific nickcan be introduced to the 5′ end of the nucleic acid strand hybridized tothe extended first primer product. In some embodiments, a nick can beintroduced into an adaptor that is linked to the proximal end of thenucleic acid strand. In some embodiments, the method can further includedegrading a portion of the nucleic acid strand, thereby generating asingle-stranded portion within the extended first primer product. Insome embodiments, a portion of the extended first primer product canremain hybridized to the nucleic acid strand. In some embodiments, themethod can further include extending the single-stranded portion withinthe extended first primer product, thereby obtaining a second sequencingread. In some embodiments, the method can include aligning the first andsecond sequencing read to obtain a nucleic acid sequence with improvedaccuracy. In some embodiments, obtaining a first and second sequencingread results in improved sequencing accuracy and/or sequencingthroughput. In some embodiments, obtaining a first and second sequencingread can include generating greater than 1 gigabyte of sequencing datain a first and second orientation. In some embodiments, obtaining afirst and second sequencing read can include generating greater than 1gigabyte of sequencing data in each of a first and second orientation.In some embodiments, obtaining a first and second sequencing read caninclude greater than 1 gigabyte of sequencing data in a firstorientation and greater than 1 gigabyte of sequencing data in a reverseorientation as compared to the first sequencing read. In someembodiments, obtaining a first and second sequencing read can includegreater than 1 gigabyte of sequencing data in a first orientation atAQ20 and greater than 1 gigabyte of sequencing data in a reverseorientation at AQ20 as compared to the first sequencing read. In someembodiments, obtaining a first and second sequencing read can includegreater than 1 gigabyte of sequencing data in a first orientation atAQ17 and greater than 1 gigabyte of sequencing data in a reverseorientation at AQ17 as compared to the first sequencing read.

In some embodiments, the sequencing can include hybridizing a firstprimer to a template strand. Optionally, the sequencing can includeextending the first primer, thereby generating an extended first primerproduct. Extending the first primer can include obtaining sequenceinformation in the “forward” or first direction. Extending the firstprimer can include using one or more enzymes. Optionally, the templatestrand can be linked to a support.

In some embodiments, the sequencing can include degrading at least someportion of the template strand. In some embodiments, the degrading canincluding degrading a portion of the template strand that is hybridizedto the extended first primer product. The degrading can include usingone or more degrading agents. In some embodiments, the degrading agentspecifically degrades the template strand but not the extended firstprimer strand, thereby generating a single-stranded region within theextended first primer product. In some embodiments, the template strandcan be degraded using a nicking enzyme, optionally in the presence of anexonuclease. The single-stranded region within the extended first primerproduct can optionally be used as a template for template-dependentnucleotide incorporation. Such template-dependent nucleotideincorporation can include catalyzing template dependent nucleotideincorporation using one or more enzymes, thereby obtaining sequenceinformation in the “reverse” or second direction. In some embodiments,template-dependent nucleotide incorporation of the single-strandedregion within the extended first primer product can include generatingan extended second nucleic acid molecule that is substantiallycomplementary to the extended first primer product over at least someportion of their respective lengths.

In some embodiments, the disclosed methods for bi-directional sequencingof nucleic acids comprise two or more sequencing reactions performedserially on the same template nucleic acid molecule. In someembodiments, the disclosed methods for bi-directional sequencing caninclude one or more hybridization reactions, extension reactions,sequencing reactions, and degradation reactions. In some embodiments,the disclosed methods for bi-directional sequencing can include a singlehybridization reaction. In some embodiments, the disclosed methods forbi-directional sequencing can be performed using a single primer.Optionally, the single primer can be hybridized to the template strandand extended by template dependent nucleotide incorporation in thepresence of a polymerase. In some embodiments, a hybridization reactioncan introduce and/or hybridize a primer to a complementary sequencealong the template strand. An extension reaction can attach orincorporate nucleotides using one or more enzymes to extend the primersequence. Such attachment or incorporation can optionally occur intemplate-dependent fashion. In some embodiments, a sequencing reactioncan determine the base identity of one or more nucleotides incorporatedinto an extending or newly synthesized nucleic acid molecule (e.g., anextending or newly synthesized primer or template strand) in anextension reaction and therefore determine sequence information relatingto the extending or newly synthesized strand. The sequencing reactioncan be performed in a forward and/or reverse direction (i.e.,bi-directional). In some embodiments, the sequencing reaction can beperformed in a first orientation, optionally followed by a secondsequencing reaction in an orientation that is reversed relative to thefirst orientation. In some embodiments, the sequencing reaction includesdetermining the identity of nucleotides incorporated during thesequencing reaction. In some embodiments, a degradation reaction caninclude the use of one of more degrading agents to degrade at least someportion of a strand of the nucleic acid molecule to be degraded. In someembodiments, the degrading can occur near a site of attachment or actionof the degrading agent. In some embodiments, the degrading reaction candegrade one or more nucleotides at one or more degradation sites alongthe nucleic acid molecule to be degraded. In some embodiments, thedegrading agent can generate one or more single-stranded regions withinthe nucleic acid molecule. In some embodiments, the single-strandedregions can be extended using one or more enzymes and optionally, theextended primer product as a template for extension. Optionally,extension of the single-stranded regions can be coupled to a sequencingreaction to determine the nucleotide identity of one or more nucleotidesincorporated into the extended nucleic acid molecule.

Methods for bi-directional sequencing of nucleic acids can be practicedon any nucleic acid, including DNA, cDNA, RNA, RNA/DNA hybrids, andnucleic acid analogs. These and other features are provided herein.

DRAWINGS

FIG. 1 is a schematic depicting an exemplary embodiment of a paired-endsequencing method according to the disclosure.

FIG. 2 is a schematic depicting an exemplary embodiment of a paired-endsequencing method according to the disclosure as compared to single-endsequencing.

FIG. 3 is a schematic depicting an exemplary embodiment of a paired-endsequencing method according to the disclosure.

FIG. 4 is a schematic depicting an exemplary embodiment of a paired-endsequencing workflow method according to the disclosure.

FIG. 5A is a graph disclosing total error rate for dual reads (2 reads)obtained using an exemplary paired-end sequencing method according tothe disclosure.

FIG. 5B is a graph disclosing consolidated reads obtained using anexemplary paired-end sequencing method according to the disclosure.

FIG. 6A is a graph disclosing total deletion rate for dual reads (2reads) obtained using an exemplary paired-end sequencing methodaccording to the disclosure.

FIG. 6B is a graph disclosing consolidated reads obtained using anexemplary paired-end sequencing method according to the disclosure.

FIG. 7A is a graph disclosing total insertion rate for dual reads (2reads) obtained using an exemplary paired-end sequencing methodaccording to the disclosure.

FIG. 7B is a graph disclosing total insertion rate for consolidatedreads obtained using an exemplary paired-end sequencing method accordingto the disclosure.

FIG. 8A is a graph disclosing total mismatch rate for dual reads (2reads) obtained using an exemplary paired-end sequencing methodaccording to the disclosure.

FIG. 8B is a graph disclosing total mismatch rate consolidated readsobtained using an exemplary paired-end sequencing method according tothe disclosure.

FIG. 9 discloses dual (2 reads) and consolidated reads obtained using anexemplary paired-end sequencing method according to the disclosure froma mapped region of the genome. An overall improvement in accuracy isobtained using the exemplary paired-end sequencing method according tothe disclosure.

FIG. 10A is a graph disclosing total error rate for dual reads (2 reads)obtained using an exemplary paired-end sequencing method according tothe disclosure.

FIG. 10B is a graph disclosing total error rate for consolidated readsobtained using an exemplary paired-end sequencing method according tothe disclosure.

FIG. 11A is a graph disclosing total deletion rate for dual reads (2reads) obtained using an exemplary paired-end sequencing methodaccording to the disclosure.

FIG. 11B is a graph disclosing total deletion rate for consolidatedreads obtained using an exemplary paired-end sequencing method accordingto the disclosure.

FIG. 12A is a graph disclosing total insertion rate for dual reads (2reads) obtained using an exemplary paired-end sequencing methodaccording to the disclosure.

FIG. 12B is a graph disclosing total insertion rate for consolidatedreads obtained using an exemplary paired-end sequencing method accordingto the disclosure.

FIG. 13A is a graph disclosing total mismatch rate for dual reads (2reads) obtained using an exemplary paired-end sequencing methodaccording to the disclosure.

FIG. 13B is a graph disclosing total mismatch rate for consolidatedreads obtained using an exemplary paired-end sequencing method accordingto the disclosure.

FIG. 14 is a schematic depicting an exemplary embodiment of a paired-endsequencing method according to the disclosure.

FIG. 15A provides data obtained using an exemplary paired-end sequencingmethod according to the disclosure.

FIG. 15B provides data obtained using an exemplary paired-end sequencingmethod according to the disclosure.

FIG. 16A provides data obtained using an exemplary paired-end sequencingmethod according to the disclosure.

FIG. 16B provides data obtained using an exemplary paired-end sequencingmethod according to the disclosure.

FIG. 17A provides data obtained using an exemplary paired-end sequencingmethod according to the disclosure.

FIG. 17B provides data obtained using an exemplary paired-end sequencingmethod according to the disclosure.

FIG. 18A provides data obtained using an exemplary paired-end sequencingmethod according to the disclosure.

FIG. 18B provides data obtained using an exemplary paired-end sequencingmethod according to the disclosure.

FIG. 18C provides data obtained using an exemplary paired-end sequencingmethod according to the disclosure.

FIG. 19A provides data obtained using an exemplary paired-end sequencingmethod according to the disclosure.

FIG. 19B provides data obtained using an exemplary paired-end sequencingmethod according to the disclosure.

FIG. 19C provides data obtained using an exemplary paired-end sequencingmethod according to the disclosure.

FIG. 20A provides data obtained using an exemplary paired-end sequencingmethod according to the disclosure.

FIG. 20B provides data obtained using an exemplary paired-end sequencingmethod according to the disclosure.

FIG. 21A provides data obtained using an exemplary paired-end sequencingmethod according to the disclosure.

FIG. 21B provides data obtained using an exemplary paired-end sequencingmethod according to the disclosure.

FIG. 22 provides comparative data of two reducing agents for use inexemplary paired end sequencing methods according to the disclosure.

FIG. 23A provides data obtained using an exemplary paired end sequencingmethod according to the disclosure.

FIG. 23B provides data obtained using an exemplary paired end sequencingmethod according to the disclosure.

FIG. 23C provides data obtained using an exemplary paired end sequencingmethod according to the disclosure.

FIG. 23D provides data obtained using an exemplary paired end sequencingmethod according to the disclosure.

FIG. 23E provides data obtained using an exemplary paired end sequencingmethod according to the disclosure.

FIG. 23F provides data obtained using an exemplary paired end sequencingmethod according to the disclosure.

FIG. 24 provides comparative data of oligonucleotide substrates toexonuclease digestion.

FIG. 25 provides comparative data of oligonucleotide substrates toexonuclease digestion.

FIG. 26 provides comparative data of oligonucleotide substrates toexonuclease digestion.

FIG. 27 provides comparative data of oligonucleotide substrates topolymerase extension.

DESCRIPTION

The following description of various exemplary embodiments is exemplaryand explanatory only and is not to be construed as limiting orrestrictive in any way. Other embodiments, features, objects, andadvantages of the present teachings will be apparent from thedescription and accompanying drawings, and from the claims

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of ordinary skillin the art to which these inventions belong. All patents, patentapplications, published applications, treatises and other publicationsreferred to herein, both supra and infra, are incorporated by referencein their entirety. If a definition and/or description is explicitly orimplicitly set forth herein that is contrary to or otherwiseinconsistent with any definition set forth in the patents, patentapplications, published applications, and other publications that areherein incorporated by reference, the definition and/or description setforth herein prevails over the definition that is incorporated byreference.

As used herein, the terms “comprise”, “comprises”, “comprising”,“contain”, “contains”, “containing”, “have”, “having” “include”,“includes”, and “including” and their variants are not intended to belimiting, are inclusive or open-ended and do not exclude additional,unrecited additives, components, integers, elements or method steps. Forexample, a process, method, system, composition, kit, or apparatus thatcomprises a list of features is not necessarily limited only to thosefeatures but may include other features not expressly listed or inherentto such process, method, system, composition, kit, or apparatus.

The practice of the present subject matter may employ, unless otherwiseindicated, conventional techniques and descriptions of organicchemistry, molecular biology (including recombinant techniques), cellbiology, and biochemistry, which are within the skill of the art. Suchconventional techniques include, but are not limited to, preparation ofsynthetic polynucleotides, polymerization techniques, nucleic acidamplification or purification, chemical and physical analysis of polymerparticles, nucleic acid sequencing and analysis, chemical andphoto-crosslinking of substrates, conjugation chemistry, and the like.Specific illustrations of suitable techniques can be used by referenceto the example herein below. Other equivalent conventional procedurescan also be used. Such conventional techniques and descriptions can befound in standard laboratory manuals such as Genome Analysis: ALaboratory Manual Series (Vols. I-IV), PCR Primer: A Laboratory Manual,and Molecular Cloning: A Laboratory Manual (all from Cold Spring HarborLaboratory Press), Hermanson, Bioconjugate Techniques, Second Edition(Academic Press, 2008); Merkus, Particle Size Measurements (Springer,2009); Rubinstein and Colby, Polymer Physics (Oxford University Press,2003); and the like.

The term “complementary” and its variants, as used herein with referenceto two or more nucleic acid sequences, refer to any nucleic acidsequences (e.g., portions of target nucleic acid molecules and primers)that can undergo cumulative base pairing at two or more individualcorresponding positions in antiparallel orientation, as in a hybridizedduplex. Optionally there can be “complete” or “total” complementaritybetween a first and second nucleic acid sequence where each nucleotidein the first nucleic acid sequence can undergo a stabilizing basepairing interaction with a nucleotide in the corresponding antiparallelposition on the second nucleic acid sequence (however, the term“complementary” by itself can include nucleic acid sequences that arenot completely complementary over their entire length); “partial”complementarity describes nucleic acid sequences in which at least 20%,but less than 100%, of the residues of one nucleic acid sequence arecomplementary to residues in the other nucleic acid sequence. In someembodiments, at least 50%, but less than 100%, of the residues of onenucleic acid sequence are complementary to residues in the other nucleicacid sequence. In some embodiments, at least 70%, 80%, 90% or 95%, butless than 100%, of the residues of one nucleic acid sequence arecomplementary to residues in the other nucleic acid sequence. Sequencesare said to be “substantially complementary” when at least 85% of theresidues of one nucleic acid sequence are complementary to residues inthe other nucleic acid sequence. “Noncomplementary” describes nucleicacid sequences in which less than 20% of the residues of one nucleicacid sequence are complementary to residues in the other nucleic acidsequence. A “mismatch” is present at any position in the two opposednucleotides are not complementary. Complementary nucleotides includenucleotides that are efficiently incorporated by DNA polymerasesopposite each other during DNA replication under physiologicalconditions. In a typical embodiment, complementary nucleotides can formbase pairs with each other, such as the A-T/U and G-C base pairs formedthrough specific Watson-Crick type hydrogen bonding between thenucleobases of nucleotides and/or polynucleotides at positionsantiparallel to each other. The complementarity of other artificial basepairs can be based on other types of hydrogen bonding and/orhydrophobicity of bases and/or shape complementarity between bases.

The term “hybridize” and its variants, as used herein with reference totwo or more nucleic acid molecules, refer to the process whereby anynucleic acid sequences within the two nucleic acid molecules (e.g., anyportions of target nucleic acid molecules and primers) undergocumulative base pairing at two or more individual correspondingpositions in antiparallel orientation, as in a hybridized duplex.Optionally there can be “complete” or “total” hybridization between afirst and second nucleic acid molecule where each nucleotide in thefirst nucleic acid sequence can undergo a stabilizing base pairinginteraction with a nucleotide in the corresponding antiparallel positionon the second nucleic acid sequence; however, the term “hybridize” byitself can include base pairing between nucleic acid sequences that arenot completely complementary over their entire length. “Partial”hybridization describes the process whereby two nucleic acid sequencesundergo cumulative base pairing at two or more individual correspondingpositions in antiparallel orientation, in which at least 20%, but lessthan 100%, of the residues of one nucleic acid sequence arecomplementary to residues in the other nucleic acid sequence. In someembodiments, hybridization includes base pairing between two nucleicacid sequences, where at least 50%, but less than 100%, of the residuesof one nucleic acid sequence are base paired with corresponding residuesin the other nucleic acid sequence. In some embodiments, at least 70%,80%, 90% or 95%, but less than 100%, of the residues of one nucleic acidsequence are base paired with corresponding residues in the othernucleic acid sequence. Sequences are said to be “substantiallyhybridized” when at least 85% of the residues of one nucleic acidsequence participate in cumulative at two or more individualcorresponding positions with corresponding residues in the other nucleicacid sequence in antiparallel orientation. In situations where onenucleic acid molecule is substantially longer than the other (or wherethe two nucleic acid molecule include both substantially complementaryand substantially non-complementary regions), the two nucleic acidmolecules can be described as “hybridized” even when portions of eitheror both nucleic acid molecule can remain unhybridized. “Unhybridized”describes nucleic acid sequences in which less than 20% of the residuesof one nucleic acid sequence are complementary to residues in the othernucleic acid sequence. In some embodiments, base pairing can occuraccording to some conventional pairing paradigm, such as the A-T/U andG-C base pairs formed through specific Watson-Crick type hydrogenbonding between the nucleobases of nucleotides and/or polynucleotidespositions antiparallel to each other; in other embodiments, base pairingcan occur through any other paradigm whereby base pairing proceedsaccording to established and predictable rules.

As used herein, the term “sequencing” and its variants compriseobtaining sequence information from a nucleic acid strand, typically bydetermining the identity of at least some nucleotides (including theirnucleobase components) within the nucleic acid molecule. While in someembodiments, “sequencing” a given region of a nucleic acid moleculeincludes identifying each and every nucleotide within the region that issequenced, in some embodiments “sequencing” comprises methods wherebythe identity of only some of the nucleotides in the region isdetermined, while the identity of some nucleotides remains undeterminedor incorrectly determined. Any suitable method of sequencing may beused. In an exemplary embodiment, sequencing can include label-free orion based sequencing methods. In some embodiments, sequencing caninclude labeled or dye-containing nucleotide or fluorescent basednucleotide sequencing methods. In some embodiments, sequencing caninclude cluster-based sequencing or bridge sequencing methods.

As used herein, the phrase “next generation sequencing” refers tosequencing technologies having increased throughput as compared totraditional Sanger- and capillary electrophoresis-based approaches, forexample with the ability to generate hundreds of thousands or millionsof relatively small sequence reads at a time. Some examples of nextgeneration sequencing techniques include, but are not limited to,sequencing by synthesis, sequencing by ligation, and sequencing byhybridization. Examples of next generations sequencing methods includepyrosequencing as used by 454 Corporation, Illumina's Solexa system, theSOLiD™ (Sequencing by Oligonucleotide Ligation and Detection) system(Life Technologies Inc.), and Ion Torrent Sequencing systems such as thePersonal Genome Machine or the Proton Sequencer (Life Technologies Inc).

The term “template nucleic acid”, “template polynucleotide”, “targetnucleic acid” “target polynucleotide”, “template strand” and variationsrefer to a nucleic acid strand that serves as the basis nucleic acid forgenerating a complementary nucleic acid strand. The sequence of thetemplate nucleic acid can be complementary to the sequence of thecomplementary strand. The template nucleic acid can be subjected tonucleic acid analysis, including sequencing and composition analysis.The template nucleic acids can be isolated in any form, includingchromosomal, genomic, organellar (e.g., mitochondrial, chloroplast orribosomal), recombinant molecules, cloned, amplified, cDNA, RNA such asprecursor mRNA or mRNA, oligonucleotides, whole genomic DNA, obtainedfrom fresh frozen paraffin embedded tissue, needle biopsies, cell freecirculating DNA, or any type of nucleic acid library. The target nucleicacid molecules may be isolated from any source including from organismssuch as prokaryotes, eukaryotes (e.g., humans, plants and animals),fungus, and viruses; cells; tissues; normal or diseased cells ortissues, body fluids including blood, urine, serum, lymph, tumor,saliva, anal and vaginal secretions, amniotic samples, perspiration, andsemen; environmental samples; culture samples; or synthesized nucleicacid molecules prepared using recombinant molecular biology or chemicalsynthesis methods. The template nucleic acid can be chemicallysynthesized to include any type of nucleic acid analog.

The term “complementary nucleic acid”, “complement polynucleotide”,“nucleic acid having a sequence complementary to a template strand”, andvariations refer to a nucleic acid strand that can be generated using atemplate nucleic acid as a basis nucleic acid. The complement nucleicacid can have a sequence that is complementary to the sequence of thetemplate strand. The complement nucleic acid can be subjected to nucleicacid analysis, including sequencing and composition analysis.

The terms “identity” and “identical” and their variants, as used herein,when used in reference to two or more nucleic acid sequences, refer tosimilarity in sequence of the two or more sequences (e.g., nucleotide orpolypeptide sequences). In the context of two or more homologoussequences, the percent identity or homology of the sequences orsubsequences thereof indicates the percentage of all monomeric units(e.g., nucleotides or amino acids) that are the same (i.e., about 70%identity, preferably 75%, 80%, 85%, 90%, 95% or 99% identity). Thepercent identity can be over a specified region, when compared andaligned for maximum correspondence over a comparison window, ordesignated region as measured using a BLAST or BLAST 2.0 sequencecomparison algorithms with default parameters described below, or bymanual alignment and visual inspection. Sequences are said to be“substantially identical” when there is at least 85% identity at theamino acid level or at the nucleotide level. Preferably, the identityexists over a region that is at least about 25, 50, 100, 150, or 200residues in length, or across the entire length of at least one comparedsequence. A typical algorithm for determining percent sequence identityand sequence similarity are the BLAST and BLAST 2.0 algorithms, whichare described in Altschul et al, Nuc. Acids Res. 25:3389-3402 (1977).Other methods include the algorithms of Smith & Waterman, Adv. Appl.Math. 2:482 (1981), and Needleman & Wunsch, J. Mol. Biol. 48:443 (1970),etc. Another indication that two nucleic acid sequences aresubstantially identical is that the two molecules or their complementshybridize to each other under stringent hybridization conditions.

As used herein, the term “extend”, “extending”, “extension” and itsvariants, when used in reference to a nucleic acid molecule, refers toincorporation or attachment of nucleotides to the nucleic acid molecule.Extension of a nucleic acid molecule or a primer can include attachmentor incorporation of natural nucleotides and/or nucleotide analogs. Suchextension can optionally be performed in a template-dependent fashion.Any suitable method of extending a nucleic acid molecule may be used.

The concept of label-free nucleic acid sequencing, including ion-basednucleic acid sequencing, is described in more detail in the followingreferences: Rothberg et al, U.S. Patent Publication Nos. 2009/0026082,2009/0127589, 2010/0301398, 2010/0300895, 2010/0300559, 2010/0197507,and 2010/0137143, which are incorporated by reference herein in theirentireties. Briefly, in such nucleic acid sequencing applications,nucleotide incorporations are determined by detecting the presence ofnatural byproducts of polymerase-catalyzed nucleic acid synthesisreactions, including hydrogen ions, polyphosphates, PPi, and Pi (e.g.,in the presence of pyrophosphatase).

In a typical embodiment of ion-based nucleic acid sequencing, nucleotideincorporations are detected by detecting the presence and/orconcentration of hydrogen ions generated by polymerase-catalyzed nucleicacid synthesis reactions, including for example primer extensionreactions. In one embodiment, templates that are operably bound to aprimer and a polymerase and that are situated within reaction chambers(such as the microwells disclosed in Rothberg et al, cited above), aresubjected to repeated cycles or flows of polymerase-catalyzed nucleotideaddition to the primer (“adding step”) followed by washing (“washingstep”). In some embodiments, such templates may be attached as clonalpopulations to a solid support, such as a microparticle, bead, or thelike, and said clonal populations are loaded into reaction chambers. Asused herein, “operably bound” means that a primer is annealed to atemplate so that the primer can be extended by a polymerase and that apolymerase is bound to such primer-template duplex, or in closeproximity thereof so that primer extension takes place whenevernucleotides are supplied.

In each adding step of the cycle, the polymerase extends the primer byincorporating added nucleotide in a template-dependent fashion, suchthat the nucleotide is incorporated only if the next base in thetemplate is the complement of the added nucleotide. If there is onecomplementary base, there is one incorporation, if two, there are twoincorporations, if three, there are three incorporations, and so on.With each such incorporation there is a hydrogen ion released, andcollectively a population of templates releasing hydrogen ions changesthe local pH of the reaction chamber. In some embodiments, theproduction of hydrogen ions is proportional to (e.g., monotonicallyrelated) to the number of contiguous complementary bases in the template(as well as the total number of template molecules with primer andpolymerase that participate in an extension reaction). Thus, when thereare a number of contiguous identical complementary bases in the template(i.e. a homopolymer region), the number of hydrogen ions generated, andtherefore the magnitude of the local pH change, is proportional to thenumber of contiguous identical complementary bases. If the next base inthe template is not complementary to the added nucleotide, then noincorporation occurs and no hydrogen ion is released. In someembodiments, after each step of adding a nucleotide, a washing step isperformed, in which an unbuffered wash solution at a predetermined pH isused to remove the nucleotide of the previous step in order to preventmisincorporations in later cycles. In some embodiments, after each stepof adding a nucleotide, an additional step may be performed wherein thereaction chambers are treated with a nucleotide-destroying agent, suchas apyrase, to eliminate any residual nucleotides remaining in thechamber, thereby minimizing the probability of spurious extensions insubsequent cycles. In some embodiments, the treatment may be included aspart of the washing step itself.

In one exemplary embodiment, different kinds (or “types”) of nucleotidesare added sequentially to the reaction chambers, so that each reactionis exposed to the different nucleotide types one at a time. For example,nucleotide types can be added in the following sequence: dATP, dCTP,dGTP, dTTP, dATP, dCTP, dGTP, dTTP, and so on; with each exposurefollowed by a wash step. The cycles may be repeated for 50 times, 100times, 200 times, 300 times, 400 times, 500 times, 750 times, or more,depending on the length of sequence information desired. In someembodiments, nucleotides can be added to the reaction chamber in a knownorder. In some embodiments, nucleotides can be added to the reactionchamber in a fixed (repeating cycle) or random order, optionally wherethe identity of each nucleotide is known prior to addition to thereaction chamber.

In some embodiments, the disclosed methods can be used to providebi-directional sequencing of a template strand in an ion-basedsequencing system, such as the Ion Torrent PGM™ sequencer or ProtonSequencer (Life Technologies, Carlsbad, Calif.). In some embodiments,sequencing accuracy can be improved according to methods, compositions,kits and apparatuses of the disclosure. For example, sequencing datausing exemplary paired end sequencing methods according to thedisclosure result in increased sequencing accuracy as compared to singleend sequencing reactions. While not wishing to be bound or limited bythe following, it is proposed that paired end sequencing reactions allowfor increased accuracy by consolidating the data obtained from both theforward and reverse reads (FIG. 4). Consolidation of paired end data(i.e., fastq x and fastq y) allows for the identification of nucleotideincorporation which is readily resolved by comparing the nucleotideincorporation data of the forward read with the overlapping reverseread. The issue of sequencing accuracy becomes more prominent as apolymerase moves further away from the sequencing start (or initiation)site. Polymerases are inherently more likely to mis-incorporate anucleotide or dissociate from the nucleic acid strand to be sequenced asthe distance from the start site increases, particularly once thedistance reaches one hundred or more nucleotides. Thus, the paired-endsequencing methods, kits, processes and compositions according to thedisclosure are well suited for both short (less than 200 base pairs) andlong read (i.e., greater than 200 base pairs, 300 base pairs, 400 basepairs, 500 base pairs, and longer) nucleic acid sequencing. In someembodiments, the disclosure generally relates to methods, compositions,kits, systems and apparatuses for improving nucleic acid sequencingaccuracy. In some embodiments, the disclosure generally relates tomethods, compositions, kits, systems and apparatuses for improving longread nucleic acid sequencing accuracy. For example, in some embodimentsthe disclosure relates generally to a method for sequencing a templatestrand in both directions using an ion-based sequencing system,comprising contacting a template strand with a first primer that isattached to a support (also referred to herein as a “support-linkedfirst primer”). The first support-linked primer can include a sequencethat is substantially complementary to a corresponding sequence in thenucleic acid template. The contacting can be performed under hybridizingconditions, such that the template strand hybridizes to thesupport-linked first primer. Typically, the first primer is linked tothe support at or near the 5′ end, while the 3′ end of thesupport-linked first primer remains available for hybridization and/ortemplate-dependent extension by a polymerase. The support-linked firstprimer optionally hybridizes to the nucleic acid template at or near the3′ end of the template. The method can further include extending thesupport-linked first primer in a template-dependent fashion, therebyforming an extended first primer product (EFPP), which is also linked tothe support. The extended first primer product typically includes asequence that is substantially complementary to a sequence of thetemplate strand, and the extended first primer product can be hybridizedto the template strand to form a first nucleic acid duplex.

The method can further include removing the template via melting ordegradation. In some embodiments, the first primer (and as a result, theextended first primer product) is linked to the support, such thatmelting or degradation of the duplex formed by the template and theextended first primer product allows removal and separation of thetemplate strand, generating an extended first primer product that islinked to the support.

In some embodiments, the support-linked first primer includes a nickingsite that can be recognized and nicked by a nicking agent. Followingextension of the first primer, the nicking site will be included in theresulting extended first primer product.

In some embodiments, the method further includes contacting thesupport-linked extended first primer product with a second primer underhybridizing conditions so that the second primer can hybridize to theextended first primer product. The second primer optionally hybridizesto the extended first primer product at or near the 3′ end of theextended first primer product. In some embodiments, the method furtherincludes extending the second primer using a polymerase, thereby formingan extended second primer product (ESPP). In a typical embodiment, thesecond primer is extended towards the support, thereby generating anESPP whose 3′ end points towards the surface of the support.

Optionally, extending the second primer can include sequencing bysynthesis. For example, the extending can include detectingincorporation of each (or some) of the nucleotides during the extension,and/or determining the identity of each (or some) of the nucleotidesduring the extension. In a typical embodiment, the nucleic acid duplexis bound to the support within a microwell of an ion-based sequencingsystem (e.g., Ion Torrent PGM™ system), where the well is operationallyassociated with a FET capable of sensing the presence of nucleotideincorporation byproducts. The four different nucleotide types (A, C, Gand T) are each contacted serially with the second primer undernucleotide polymerization conditions; only nucleotides that arecomplementary to the next base in the extended first primer product(EFPP) will be incorporated into the second primer by the polymerase,and such nucleotide incorporation is detecting by detecting the presenceof nucleotide incorporation byproducts (e.g., hydrogen ions) using theFET to obtain a first sequencing read.

In some embodiments, the EFPP includes a nicking site that can berecognized and nicked by a nicking agent. For example, the nicking sitecan be present in the first primer itself, such that following extensionof the first primer, the nicking site will be included in the resultingextended first primer product. Typically, the second primer is extendedpast the nicking site within the EFPP, so that the ESPP will includesequence that is complementary to the nicking site in the EFPP.

The method can further include nicking the extended first primer productat the nicking site using a suitable nicking agent. In some embodiments,the nicking agent can include one or more nickases. In some embodiments,the nicking agent can include one or more site-specific nickases orrestriction enzymes. In some embodiments, the nicking agent canspecifically nick the template strand while leaving the extended firstprimer product or complementary strand unaffected.

The method can further include degrading the EFPP using a suitabledegrading agent. In a typical embodiment, the degrading includescontacting a nucleic acid duplex including the nicked EFPP hybridized tothe ESPP with a 5′ to 3′ exonuclease, and degrading a portion of theEFPP from the 5′ end of the nick via 5′ to 3′ exonuclease digestion,while leaving a portion (“residual portion”) of the EFPP substantiallyintact or undegraded. This residual portion typically includes the 3′end of the nick plus associated “upstream” sequence that is linked at ornear the primer, and remains hybridized to a portion of an ESPP. Suchdigestion can render a portion of the ESPP single-stranded. A portion ofthe ESPP can remain hybridized to the residual portion of the EFPP.

In embodiments where the extending includes ion-based sequencing bysynthesis, the nicking agent and/or the degrading agent can be addeddirectly to the ion based sequencing system. For example, in someembodiments, nicking enzymes and degrading agents can be added directlyto the Ion chip within a PGM™ sequencer. Alternatively, the Ion Chip canbe placed in a Paired-End module configured to allow such reagentexchange during the sequencing.

In a typical embodiment, the degrading does not affect the ESPP becausethe ESPP is modified or treated so that it is resistant to thedegrading. For example, in some embodiments the second primer used tosynthesize the ESPP can be resistant to digestion by the degradingagent, so that the ESPP is not digested by the degrading agent.

In some embodiments, the method can further include extending theresidual portion of the EFPP after the degrading. The residual portionof the EFPP remains hybridized to the ESPP and therefore can be extendedin a template-dependent manner. In some embodiments, extending theresidual portion of the EFPP can include sequencing by synthesis,thereby obtaining a second sequencing read in the opposite directionfrom the first. Typically, the extending can include the use of one ormore polymerases such as, but not limited to Klenow, DNA polymerase I,and T4 DNA polymerase. For example, the extending can be performed in amicrowell of an ion-based sequencing system (e.g., Ion Torrent PGM™sequencer and Proton™ sequencer) and include detecting incorporation ofeach (or some) of the nucleotides during the extension, and/ordetermining the identity of each (or some) of the nucleotides during theextension using the PGM™ or Proton™ sequencer. In some embodiments, aremoval or wash step is carried out prior to extension of the residualportion of the EFPP but after extension of the extended first primerproduct.

In some embodiments, the template strand is directly linked to thesupport or surface, such that there is no need for hybridization to asupport-linked primer in order to link the template (or its complement)to the support or surface. In such embodiments, the process can besimplified to include only two separate primer extension steps, ratherthan the three steps described above.

In some embodiments, any one or more of the nucleic acid moleculesreferred to herein (including without limitation the first primer, thesecond primer, the template strand, the first primer extension productand the second primer extension product) can be linked, or can bemodified to support linkage, to a surface or solid support. For example,in some embodiments the nucleic acid molecule can be linked to onemember of a binding pair, while the surface or support can be linked tothe other member of the binding pair. As used herein, the term “bindingpair” and its variants refers to two molecules, or portions thereof,which have a specific binding affinity for one another and typicallywill bind to each other in preference to binding to other molecules.Typically but not necessarily some or all of the structure of one memberof a specific binding pair is complementary to some or all of thestructure possessed by the other member, with the two members being ableto bind together specifically by way of a bond between the complementarystructures, optionally by virtue of multiple noncovalent attractions.The two members of a binding pair are referred to herein as the “firstmember” and the “second member” respectively.

The following may be mentioned as non-limiting examples of moleculesthat can function as a member of a specific binding pair, without thisbeing understood as any restriction: thyroxin-binding globulin,steroid-binding proteins, antibodies, antigens, haptens, enzymes,lectins, nucleic acids, repressors, oligonucleotides, polynucleotides,protein A, protein G, avidin, streptavidin, biotin, complement componentC1q, nucleic acid-binding proteins, receptors, carbohydrates,complementary nucleic acid sequences, and the like. Examples of specificbinding pairs include without limitation: an avidin moiety and a biotinmoiety; an antigenic epitope and an antibody or immunologically reactivefragment thereof; an antibody and a hapten; a digoxigen moiety and ananti-digoxigen antibody; a fluorescein moiety and an anti-fluoresceinantibody; an operator and a repressor; a nuclease and a nucleotide; alectin and a polysaccharide; a steroid and a steroid-binding protein; anactive compound and an active compound receptor; a hormone and a hormonereceptor; an enzyme and a substrate; an immunoglobulin and protein A;and an oligonucleotide or polynucleotide and its correspondingcomplement.

As used herein, the term “biotin moiety” and its variants comprisesbiotin (cis-hexahydro-2-oxo-1H-thieno[3,4]imidazole-4-pentanoic acid)and any derivatives and analogs thereof, including biotin-likecompounds. Such compounds include, for example, biotin-e-N-lysine,biocytin hydrazide, amino or sulfhydryl derivatives of 2-iminobiotin andbiotinyl-∈-aminocaproic acid-N-hydroxysuccinimide ester,sulfosuccinimideiminobiotin, biotinbromoacetylhydrazide, p-diazobenzoylbiocytin, 3-(N-maleimidopropionyl)biocytin, and the like. “Biotinmoiety” also comprises biotin variants that can specifically bind to anavidin moiety.

The term “biotinylated” and its variants, as used herein, refer to anycovalent or non-covalent adduct of biotin with other moieties such asbiomolecules, e.g., proteins, nucleic acids (including DNA, RNA, DNA/RNAchimeric molecules, nucleic acid analogs and peptide nucleic acids),proteins (including enzymes, peptides and antibodies), carbohydrates,lipids, etc.

The terms “avidin” and “avidin moiety” and their variants, as usedherein, comprises the native egg-white glycoprotein avidin, as well asany derivatives, analogs and other non-native forms of avidin, that canspecifically bind to biotin moieties. In some embodiments, the avidinmoiety can comprise deglycosylated forms of avidin, bacterialstreptavidins produced by selected strains of Streptomyces, e.g.,Streptomyces avidinii, to truncated streptavidins, and to recombinantavidin and streptavidin as well as to derivatives of native,deglycosylated and recombinant avidin and of native, recombinant andtruncated streptavidin, for example, N-acyl avidins, e.g., N-acetyl,N-phthalyl and N-succinyl avidin, and the commercial productsExtrAvidin®, Captavidin®, Neutravidin® and Neutralite Avidin®. All formsof avidin-type molecules, including both native and recombinant avidinand streptavidin as well as derivatized molecules, e.g. nonglycosylatedavidins, N-acyl avidins and truncated streptavidins, are encompassedwithin the terms “avidin” and “avidin moiety”. Typically, but notnecessarily, avidin exists as a tetrameric protein, wherein each of thefour tetramers is capable of binding at least one biotin moiety.

As used herein, the term “biotin-avidin bond” and its variants refer toa specific linkage formed between a biotin moiety and an avidin moiety.Typically, a biotin moiety can bind with high affinity to an avidinmoiety, with a dissociation constant (K_(d)) typically in the order of10⁻¹⁴ to 10⁻¹⁵ mol/L. Typically, such binding occurs via non-covalentinteractions.

For example, a nucleic acid molecule can be amino-modified forattachment to a surface or support (e.g., a microparticle or a planarsurface). In some embodiments, an amino-modified nucleic acid moleculecan be attached to a surface that is coated with a carboxylic acid. Insome embodiments, an amino-modified nucleic acid can be reacted with EDC(or EDAC) for attachment to a carboxylic acid coated surface (with orwithout NHS).

In some embodiments, a nucleic acid molecule can be modified to attachto one member of a binding pair (e.g., biotin), and thus bind to asurface or support including a second member of the binding pair. Insome embodiments, a biotinylated nucleic acid molecule can be attachedto another member of a binding pair (e.g., avidin-like, such asstreptavidin) which is attached to a surface or support. In someembodiments, the template strand can be linked to a solid support. Insome embodiments, the support can be an array, sphere, particle,microparticle, filter, gel or bead. In some embodiments, the particlecan be an Ion Sphere™ Particle (Life Technologies, CA). In someembodiments, the support can be a planar surface such as a slide, grooveor channel. In some embodiments, the support can be concave, convex, orany combination thereof, such as an array surface or plurality offlowcells. In some embodiments, the support can be a reaction chamber ormicrowell. In some embodiments, the support can include a surface with atexture such as an etching or passivation layer. In some embodiments, asupport can be made from materials such as glass, borosilicate glass,silica, quartz, fused quartz, mica, polyacrylamide, plastic polystyrene,polycarbonate, polymethacrylate (PMA), polymethyl methacrylate (PMMA),polydimethylsiloxane (PDMS), silicon, germanium, graphite, ceramics,silicon, semiconductor fabrics, high refractive index dielectrics,crystals, gels, polymers, or films (e.g., films of gold, silver,aluminum, or diamond). In some embodiments, template nucleic acidslinked to a support, optionally linked through one or more primers, canbe arranged in a random or ordered array on the support.

In some embodiments, the support can be modified to enhance attachmentof a nucleic acid molecule to the support and/or enhance sequencingthroughput from a nucleic acid molecule attached or operably bound tothe support. For example, the support can be modified to include aplurality of primers attached to its surface that are operably bound toat least some portion of a template nucleic acid molecule to besequenced (e.g., FIG. 2, Primer B). In some embodiments, the support canbe modified to include a plurality of primers partially-embedded withinthe support that are operably bound to at least some portion of atemplate nucleic acid molecule to be sequenced. In some embodiments, thesupport can be modified to include a plurality of primers anchored tothe outer surface of the support and operably bound to at least someportion of a template nucleic acid molecule to be sequenced (e.g., FIG.3, Primer B). In some embodiments, the support can include a particlewhich can optionally include a porous, permeable or scaffolded particle.

In some embodiments, a plurality of primers can be attached orimmobilized to a support at their 5′ end. In some embodiments, the 5′end of one or more of the plurality of primers attached or immobilizedto the support can be modified to enhance sequencing throughput and/oraccuracy. For example, the 5′ end of one or more primers immobilized ona support can be modified to enhance resistance to enzymaticdegradation. In some embodiments, the 5′ end of one or more primersimmobilized on a support can have enhanced resistance to exonucleaseactivity. For example, the 5′ end of one or more primers immobilized ona support can have enhanced resistance to exonuclease activity ascompared to the 5′ end of a comparable non-modified immobilized primer.In some embodiments, the 5′ end of one or more primers immobilized on asupport can be modified to protect the 5′ end of the primer fromdeterioration which can lead to template loss or reduction of sequencingthroughput when conducting “reverse” reads. In some embodiments, the 5′end of one or more primers immobilized on a support can be modified toinclude one or more phosphorothioates. For example, a primer immobilizedon a support can include one, two, three, four, five, six, seven or morephosphorothioates. In some embodiments, the 5′ end of one or moreprimers immobilized on a support can be modified to include apolyethylene glycol (PEG) linker. In some embodiments, the 5′ end of oneor more primers immobilized on a support can be modified to include asingle stranded region of about 5 nucleotides to about 15 nucleotides.In some embodiments, the 5′ end of one or more primers immobilized on asupport can be modified to include a hairpin structure. In someembodiments, the 5′ end of one or more primers immobilized on a supportcan be modified to include an abasic site. In some embodiments, the 5′end of one or more primers immobilized on a support can be modified toinclude one or more locked nucleic acids. The phrase “locked nucleicacid” (LNA) as used herein refers to a modified RNA nucleotide in whichthe ribose is modified with an extra bridge connecting the 2′ oxygen and4′ carbon. A locked nucleic acid can be resistant to cleavage byExonuclease III. In some embodiments, the 5′ end of one or more primersimmobilized on a support can be modified to include one or more 2′-OMeRNA residues. 2′-OMe RNA residues possess a methyl group at the 2′-OHresidue of the ribose molecule that protects against nucleasedegradation.

In some embodiments, the 5′ end of one or more primers immobilized on asupport can include one or more of a single stranded region of about 5nucleotides to about 15 nucleotides, a phosphorothioate, a PEG linker, ahairpin structure, an abasic site, a 2′-OMe residue or a combinationthereof.

In some embodiments, a template strand can be directly attached to asupport. In some embodiments, one or more primers (e.g., FIG. 2, PrimerB) can attach or link the template strand or the extended primer productto the support. In some embodiments, a primer attached to the supportcan be linked to the template strand by a ligase. In some embodiments,one or more primers attached to the support can contain a modificationsuch as a photolabile group that allows attachment of the templatestrand or the extended primer product to the support. In someembodiments, an extended primer product can be photo-crosslinked to thesupport, while the template strand is degraded, thereby generating asingle-stranded extended primer product attached to the support. In someembodiments, a primer attached to the support can include an aminatedresidue.

In some embodiments, the disclosure relates generally to methods,compositions, systems, apparatuses and kits for obtaining sequenceinformation from a nucleic acid molecule. Optionally, such sequenceinformation can be obtained in one or both directions (e.g., a “forward”and/or “reverse” direction) from a nucleic acid strand. In someembodiments, sequence information can be obtained in a firstorientation, optionally followed by obtaining sequencing information ina second orientation that is reversed relative to the first orientation.In some embodiments, sequence information can be obtained in one or bothdirections from a template strand. In some embodiments, such sequencingcan be referred to as “bi-directional” sequencing. Bi-directionalsequencing can optionally include sequencing at least some portion ofeach strand of a nucleic acid duplex. In some embodiments, sequenceinformation can be first obtained as a “forward” read which can includesequencing from the distal end of a support bound template nucleic acidmolecule. In some embodiments, a second or serial sequencing reactionscan be performed to obtain a “reverse” read of the initial support boundnucleic acid molecule, which can optionally include sequencing from theproximal end. In some embodiments, bi-directional sequencing can beperformed as a distal sequencing reaction followed by a proximalsequencing reaction. In some embodiments, one or more of the “forward”or “reverse” sequencing reactions can be repeated one or more times toimprove sequencing throughput and/or accuracy. For example, in someembodiments a “forward” read can be repeated by degrading or separatingan extended primer product from the template nucleic acid molecule andre-applying and hybridizing a first primer to the template nucleic acidmolecule, followed by primer extension. In some embodiments, theseparating can include a denaturing treatment such as, but not limitedto, an enzymatic, thermal or chemical degradation. In some embodiments,the extended primer product can be separated from the template nucleicacid molecule by treatment with sodium hydroxide. In some embodiments,the extended primer product can be separated from the template nucleicacid molecule by heat. In some embodiments, the “forward” read can berepeated as desired, by re-applying and hybridizing a sequencing primerto the nucleic acid molecule to be sequenced. The re-applied sequencingprimer can be extended, for example under polymerization conditions inthe presence of a polymerase and dNTPs.

In some embodiments a “reverse” read can be repeated by degrading orseparating the template nucleic acid molecule (that is substantiallycomplementary to the extended first primer product) to produce one ormore single-stranded regions within the extended first primer product.In some embodiments, the separating can include an enzymatic, thermal orchemical treatment. In some embodiments, the degrading can include oneor more enzymes that remove, digests, or nicks one or more nucleotidesincorporated into the nucleic acid molecule that is substantiallycomplementary to the extended first primer product. In some embodiments,the one or more degrading enzymes can include a nicking enzyme,optionally in combination with an exonuclease. In some embodiments, thedegrading can further include an endonuclease. In some embodiments,bi-directional sequencing of a template nucleic acid molecule canoptionally be performed as a first proximal sequencing reaction coupledwith a second distal sequencing reaction. In some embodiments,bi-directional sequencing of a template nucleic acid molecule canoptionally be performed as a first distal sequencing reaction coupledwith a second proximal sequencing reaction.

In some embodiments, the disclosure relates generally to obtainingsequence information from a nucleic acid strand, comprising: hybridizingan existing nucleic acid molecule (frequently referred to as a “primer”)to the nucleic acid strand to be sequenced (frequently referred to asthe “template”), and extending the primer via template-dependentnucleotide incorporation using a polymerase. In some embodiments, theprimer can be extending under polymerization conditions using natural oranalog nucleotides, including nucleotides having a label or fluorescentproprieties. In some embodiments, the primer can be extended underpolymerization conditions using nucleotides that are label-free. In someembodiments, sequencing information can be obtained by sequencing atleast some portion of the extended first primer product. In someembodiments, sequencing information can be obtained for substantiallyall of the extended first primer product.

In some embodiments, a first primer (also frequently referred to as a“forward” primer) is hybridized to the template strand and extended,thereby obtaining sequence information in the “forward” direction(frequently referred to as a “forward” read) and generating an extendedfirst primer molecule (frequently referred to herein as the “complement”or “extended first primer product”) that is substantially complementaryto the template strand over at least some portion of their respectivelengths. In some embodiments, the phrase “substantially complementary tothe template strand” when used in reference to an extended primerproduct refers to a situation where at least 85% of the residues of theextended primer product are complementary to the template strand. Insome embodiments, the forward primer can be fully extended (to generatea fully extended primer product) when hybridized to a nucleic acidmolecule in the presence of a polymerase and dNTPs. In some embodiments,some or all of the nucleotides incorporated into the extended firstprimer product are sequenced to provide sequence information.

It is well known in the art that extension of a nucleic acid moleculegenerally includes contacting the nucleic acid molecule with apolymerase and nucleotides, under nucleotide incorporation conditions.Generally, a polymerase is bound to, or is in close proximity to, thenucleic acid molecule to facilitate attachment or incorporation ofnucleotides to the nucleic acid molecule. Typically, a polymeraseextends the nucleic acid molecule by incorporating a nucleotide if thenext base in the nucleic acid molecule is the complement of the addednucleotide. If there is one complementary base, there is oneincorporation, if two, there are two incorporations, if three, there arethree incorporations, and so on. In an exemplary embodiment, thepolymerase includes any enzyme, or fragment or subunit thereof, whichcan catalyze incorporation of nucleotides and/or nucleotide analogs. Insome embodiments, extension reactions can be conducted using a DNA orRNA polymerase enzyme. In some embodiments, the DNA polymerase can be athermostable polymerase. In some embodiments, a polymerase can include ahigh fidelity polymerase. In an exemplary embodiment, a polymerase canbe a naturally-occurring polymerase, recombinant polymerase, mutantpolymerase, variant polymerase, fusion or otherwise engineeredpolymerase, chemically modified polymerase, synthetic molecules, oranalog, derivative or fragment thereof. In some embodiments, athermostable polymerase includes a recombinant polymerase, mutantpolymerase, variant polymerase, fusion or otherwise engineeredpolymerase, chemically modified polymerase, derivative or fragment ofone or more of the following thermostable polymerases: Taq polymerase(from Thermus aquaticus), Tfi polymerase (from Thermus filiformis), Pfupolymerase (from Pyrococcus furiosus), Tth (from Therms thermophilus),Pow polymerase (from Pyrococcus woesei), Tli polymerase (fromThermococcus litoralis), Pol I and II polymerases (from Pyrococcusabyssi), Pab (from Pyrococcus abyssi), Bst polymerase (from Bacillusstearothermophilus), Tli polymerase, 9° N polymerase, and phi29polymerase.

Typical conditions for nucleic acid extension can include reactionsconditions of about 25° C.-80° C. In some embodiments, extension caninclude modulating the extension conditions. Modulating can optionallyinclude: increasing or decreasing the enzyme concentration; increasingor decreasing the nucleotide concentration; increasing or decreasing acation concentration; increasing or decreasing a reaction temperature,reaction time and/or pH, and the like. The modulating can includeincreasing or decreasing the rate of the reaction, increasing ordecreasing the yield of product of the reaction, and the like. In someembodiments, extension reactions can optionally include a sequencingreaction thereby obtaining sequence information from a nucleic acidstrand, typically by determining the identity of at least somenucleotides within the nucleic acid molecule being extended.

In some embodiments, extension can be performed in the presence ofappropriate buffers and/or nucleotides (including nucleotide analogs orbiotinylated nucleotides). In some embodiments, an appropriate buffercan optionally include a detergent and/or an additive. For example, anappropriate buffer can optionally include one or more detergents suchas, but not limited to, Tween, SDS, Triton, and the like. In someembodiments, an additive can include a polymer compound comprising ahomo-polymeric and hetero-polymeric compound. In some embodiments, apolymer compound comprises a chain of two or more tetrahydrapyrrolemonomers. In some embodiments, a tetrahydrapyrrole monomer comprises afive-membered heterocyclic ring. In some embodiments, in a chain oftetrahydropyrrole rings, one or more tetrahydropyrrole rings comprise anitrogen atom reacted with a carbonyl or carboxylic acid compound. Insome embodiments, a polymer compound comprises polyvinylpyrrolidone(e.g., povidone or crospovidone), poly(4-vinylphenol), andvinylpyrrolidone/vinyl acetate copolymer (e.g., copovidone). In someembodiments, a polymer compound comprises two or more monomers ofN-vinyl-pyrrolidone, including modified polymers thereof. Modifiedpolymers of poly(N-vinyl-pyrrolidone) comprise monofunctionalized (e.g.,hydroxyl or carboxy end group), side-chain conjugates (e.g., poly- andmultifunctional side chains), and grafted copolymers. In someembodiments, polyvinylpyrrolidone includes various molecular weightpolymers including average molecular weights of about 5 kD-55 kD, forexample 10 kD, 29 kD, 40 kD, and 55 kD molecular weight compounds. Insome embodiments, an additive such as polyvinylpyrrolidone (PVP) can bepresent in an appropriate buffer at about 0.1-8%, or about 1-2%, orabout 2-3%, or about 3-4%, or about 4-5%, or about 5-6%, or about 6-7%,or about 8-10%.

In some embodiments, an appropriate buffer can include one or morereducing agents such as, but not limited to, dithiothreitol (DTT),tris(2-carboxyethyl)phosphine (TCEP), and the like. In some embodiments,a reducing agent such as DTT or TCEP can be present in an appropriatebuffer at about 0.1-8%, or about 1-2%, or about 2-3%, or about 3-4%, orabout 4-5%, or about 5-6%, or about 6-7%, or about 8-10%.

In some embodiments, the methods, kits, compositions and apparatuses ofthe disclosure further include sequencing the extended first primerproduct. Such sequencing can optionally include hybridizing a secondprimer to the extended first primer product and extending the secondprimer via template-dependent nucleotide incorporation using apolymerase, thereby obtaining sequence information in the “reverse”direction (frequently referred to as a “reverse” read of the template)and generating an extended second primer molecule (frequently referredto herein as the “template copy” or “extended second primer product”)that is substantially identical to the template strand over at leastsome portion of their respective lengths. In some embodiments,sequencing information of the reverse read can be obtained by sequencingat least some portion of the extended second primer product. In someembodiments, sequencing information of the reverse read can be obtainedfor substantially all of the extended second primer product.

In some embodiments, the methods of the disclosure further includeintroducing at least one nick or gap into one or more nucleic acidmolecules selected from the group consisting of: the first primer, thesecond primer, the template strand, the first extended primer productand the second extended primer product. As used herein, the term “nick”and its variants refers to any discontinuity in a nucleic acid strandwhere there is no phosphodiester bond between any two adjacentnucleotides of the strand.

The nick can include one free 3′ end and one free 5′ end. In someembodiments, the nicking includes introducing a nick including a free 5′end and a free 3′ end into the template strand. In some embodiments, thefree 5′ end can include a 5′ phosphate group. The free 3′ end canoptionally include a hydroxyl group.

In some embodiments, the nicking includes contacting a nucleic acidduplex and nicking one or both strands of the duplex. For example, thenicking can include contacting a duplex including the template strandhybridized to the extended first primer product with a nicking agentunder nicking conditions, thereby introducing one or more nicks into atleast one strand of the duplex. In some embodiments, the introducingincludes nicking one strand of the duplex, but not both strands of theduplex. For example, in some embodiments the template strand is nickedwhile the extended first primer product is not nicked. In someembodiments, both strands of the duplex are nicked, but are differentpositions along each strand, such that there is no double-stranded breakintroduced into the duplex.

In some embodiments, the nicking includes using a nicking agent tointroduce nicks at random and/or multiple positions within a nucleicacid molecule. In some embodiments, the nicking includes using a nickingagent to introduce nicks at defined and preselected sites within thenucleic acid molecule, for example within a specific sequence(site-specific nicking). In some embodiments, a nicking agent caninclude an enzyme, light or chemical compound. For example,site-specific nicking can be performed using a site-specific nickingenzyme.

Any suitable method of nicking a nucleic acid molecule may be used.Methods of nicking nucleic acid molecules are well known in the art. Forexample, it is well known that nicking of nucleic acid moleculesgenerally includes contacting the nucleic acid molecule with a nickingagent under nicking conditions. For example, nicking of a nucleic acidmolecule can include nicking the nucleic acid molecule by enzymatic,photo cleaving (e.g., light) or chemical methods. In an exemplaryembodiment, nicking can be conducted using a nicking enzyme. In someembodiments, a nicking enzyme can be a naturally-occurring enzyme,recombinant enzyme, mutant enzyme, variant enzyme, fusion or otherwiseengineered enzyme, chemically modified enzyme, synthetic molecule, oranalog, derivative or fragment thereof. In some embodiments, nicking canbe coupled with an enzyme that couples a 5′→3′polymerization/degradation reaction, such as E. coli DNA polymerase I,Thermus aquaticus DNA polymerase, or T4 DNA polymerase. Typicalconditions for enzymatic nicking can include reaction temperatures ofabout 0° C.-45° C. In some embodiments, a nicking reaction can beconducted for about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20 or 30 minutes. A nicking reaction can be terminatedor slowed by increasing the temperature, decreasing the temperature,altering the pH, altering the ions present, altering the salt conditionspresent, and/or addition of a chelating agent. In some embodiments,nicking can include modulating the nicking method. Modulating canoptionally include: increasing or decreasing the nicking agentconcentration; increasing or decreasing a cation concentration;increasing or decreasing a reaction temperature, reaction time and/orpH, and the like. The modulating can include increasing or decreasingthe rate of the reaction, increasing or decreasing the yield of productof the reaction, and the like. In some embodiments, nicking can beperformed in the presence of appropriate buffers and/or nucleotides(including nucleotide analogs or biotinylated nucleotides). In someembodiments, the appropriate buffer can optionally include a detergentand/or an additive. For example, a buffer can optionally include one ormore detergents such as, but not limited to, TWEEN-20, SDS, TRITON, andthe like. In some embodiments, an additive can include a polymercompound comprising a homo-polymeric and hetero-polymeric compound. Insome embodiments, a polymer compound comprises a chain of two or moretetrahydrapyrrole monomers. In some embodiments, a tetrahydrapyrrolemonomer comprises a five-membered heterocyclic ring. In someembodiments, in a chain of tetrahydropyrrole rings, one or moretetrahydropyrrole rings comprise a nitrogen atom reacted with a carbonylor carboxylic acid compound. In some embodiments, a polymer compoundcomprises polyvinylpyrrolidone (e.g., povidone or crospovidone),poly(4-vinylphenol), and vinylpyrrolidone/vinyl acetate copolymer (e.g.,copovidone). In some embodiments, a polymer compound comprises two ormore monomers of N-vinyl-pyrrolidone, including modified polymersthereof. Modified polymers of poly(N-vinyl-pyrrolidone) comprisemonofunctionalized (e.g., hydroxyl or carboxy end group), side-chainconjugates (e.g., poly- and multifunctional side chains), and graftedcopolymers. In some embodiments, polyvinylpyrrolidone includes variousmolecular weight polymers including average molecular weights of about 5kD-55 kD, for example 10 kD, 29 kD, 40 kD, and 55 kD molecular weightcompounds. In some embodiments, an additive (e.g., polyvinylpyrrolidone(PVP)) can be present in the buffer at about 0.1-8%, or about 1-2%, orabout 2-3%, or about 3-4%, or about 4-5%, or about 5-6%, or about 6-7%,or about 7-8%. In some embodiments, the appropriate buffer can include areducing agent such as, but not limited to DTT, TCEP, and the like. Insome embodiments, nicking can include translation of the nick to a newposition along the nucleic acid molecule. In some embodiments,translation of a nick can include a nick translation reaction. Methodsfor performing nick translation reactions are known to those of skill inthe art (Rigby, P. W. et al. (1977), J. Mol. Biol. 113, 237).

In some embodiments, methods for nicking nucleic acid molecules caninclude nicking the nucleic acid molecule using a nicking enzyme. Insome embodiments, nicking enzymes include any enzyme having endonucleaseactivity, with or without exonuclease activity. In some embodiments,nicking enzymes include any enzyme that can catalyze nicking one or bothstrands of a single stranded nucleic acid molecule or of adouble-stranded nucleic acid duplex. In some embodiments, nickingenzymes include any enzyme that can catalyze introducing a nick atrandom positions in one or both strands of a double-stranded nucleicacid. In some embodiments, nicking enzymes include any enzyme that canintroduce one or more nicks at random (or nearly random) positions ineither strand of a nucleic acid duplex. In some embodiments, nickingenzymes include any enzyme that can introduce one or more nicks in anon-specific sequence manner at any position in either strand of anucleic acid duplex. In some embodiments, nucleic acid nicking enzymesinclude any wild-type or mutant deoxyribonucleases I (DNase I) enzymeisolated from any organism or tissue, or isolated as a recombinantenzyme. In some embodiments, a DNase I can be isolated from bovine. Insome embodiments, a DNase I can be isolated from pancreas.

In some embodiments, the nicking enzyme can be a DNase from a familyVirionaceae, such as genus Vibrio, which includes Vibrio vulnificus. Insome embodiments, the nicking enzyme can be a Vvn polymerase. In someembodiments, the nicking enzyme can be a DNA polymerase from Vibriocholera (Focareta and Manning 1987 Gene 53(1):31-400, or an NucMpolymerase from Erwinia chrysanthemi (Moulard 1993 Mol. Microbiol.8)4):685-695, or an Endo I polymerase from E. coli (Jekel 1995 Gene154(1):55-59, or a Dns or DnsH polymerase from Aeromonas hydrophila(Chang 1992 Gene 122(1):175-180, Dodd 1999 FEMS Microbiol. Lett.173:41-46, and Wang 2007 Nucleic Acids Research 35:584-594). In someembodiments, the nicking enzyme can be a DNase from a familyEnterobacteriaceae, such as a genus Serratia, which includes Serratiamarcescens (Benzonase™, U.S. Pat. No. 5,173,418).

In some embodiments, the nicking enzyme exhibits little or no preferencefor nicking nucleic acids at sequences having a high or low GC %content, including nucleic acids having about 0-10%, or about 10-25%, orabout 25-40%, or about 40-55%, or about 55-70%, or about 70-85%, orabout 85-100% GC % content.

In some embodiments, a site-specific nicking enzyme can be used to nickthe nucleic acid molecule. Many site-specific nicking enzymes are knownin the art. For example, New England BioLabs Inc., provides a variety ofsite-specific nicking enzymes including Nt.CviPII, Nb.BsmI, Nb.BbvCI,Nb.BsrDI, Nb.BtsI, Nt.BsmAI, Nt.BbvCI, Nt.BspQI, Nt.AlwI and Nt.BstNBI.As an example, the enzyme Nt.BbvCI nicks the sequence CC^TCAGC at thesite denoted by the carrot. This particular sequence can provides a5-base “key” sequence that indicates the start of a sequence read. Inanother example, the enzyme AscI nicks the sequence GG^CGCGCC at thesite denoted by the carrot. This particular sequence can provide a6-base “key” sequence that indicates the start of a sequence read. Inyet another example, the enzyme NotI nicks the sequence GC^GGCCGC at thesite denoted by the carrot. This particular sequence can provide a6-base “key” sequence that indicates the start of a sequence read. Inanother example, the enzyme AsiSI nicks the sequence GCGAT^CGC at thesite denoted by the carrot. This particular sequence can provide a3-base “key” sequence that indicates the start of a sequence read.

In some embodiments, the nicking enzyme can be Nt.CviPII, Nb.BsmI,Nb.BbvCI, Nb.BsrDI, Nb.BtsI, Nt.BsmAI, Nt.BbvCI, Nt.BspQI, Nt.Alwi, orNt.BstNBI. In some embodiments, the nicking enzyme is incubated atdefined temperature for defined period with the nucleic acid molecule(or duplex) to be nicked. In a typical embodiment, the nicking enzyme isincubated with the nucleic acid molecule (or duplex) for about 5 minutesto about 30 minutes at 37° C. In some embodiments, the nicking enzyme isincubated with the nucleic acid molecule for about 5 minutes to about 30minutes at about 50° C. In some embodiments, the nicking enzyme isincubated with the nucleic acid molecule for about 5 minutes to about 30minutes at about 65° C. In some embodiments, the nicking enzyme can beheat inactivated after nicking and does not require a buffer exchange orclean up step, prior to advancing the method.

In some embodiments, the one or more nucleic acid molecule (e.g., thefirst primer, the second primer, the template strand, the first extendedprimer product and the second extended primer product) can include atleast one scissile linkage which can be cleavable. In some embodiments,a scissile linkage can be cleavable with an enzyme, photochemical orchemical treatment. In some embodiments, the scissile linkage can belocated at any position within the one or more nucleic acid molecules,including at or near a terminal end or in an interior portion of the oneor more nucleic acid molecules.

In some embodiments, conditions suitable for cleaving a scissile linkagecan include a pH range of about 4-10, or about 5-9, or about 6-8. Insome embodiments, conditions suitable for cleaving a scissile linkagecan include a temperature range of about 15-60° C., or about 20-55° C.,or about 25-50° C., or about 24-45° C., or about 22-40° C., or about20-35° C.

In some embodiments, a scissile linkage can include at least onephosphorothioate linkage. In some embodiments, a phosphorothioatelinkage can be cleavable with a metal compound (Vyle 1992 Biochemistry31:3012-3018; Sontheimer 1999 Methods 18:29-37; and Mag, 1991 NucleicAcids Research 19:1437-1441). In some embodiments, a metal compound caninclude silver (Ag), mercury (Hg), copper (Cu), manganese (Mn), zinc(Zn), cadmium (Cd) or iodide (I₂). In some embodiments, aphosphorothioate linkage can be cleaved with a water-soluble salt thatprovides Ag+, Hg++, Cu++, Mn++, Zn+ or Cd+ anions. Salts that provideions of other oxidation states can be used for nucleic acid cleavage.For example, a phosphorothioate linkage can be cleaved with asilver-containing salt, such as silver nitrate (AgNO₃). Double strandedDNA substrates containing a single phosphorothioate bond at the 5′ endwere found to be susceptible to T7 gene 6 exonuclease digestion; whilecomparable substrates containing four phosphorothioates were found to beresistant under the same conditions (Nikiforov 1994 Genome Research3:285-291.

In some embodiments, a scissile linkage can include at least oneacid-labile linkage. An example of an acid-labile linkage includes aphosphoramidate linkage. In some embodiments, a phosphoramidate linkagecan be hydrolysable under acidic conditions, including mild acidicconditions (Shchepinov 2001 Nucleic Acids Research 29:3864-3872; Mag1992 Tetrahedron Letters 33:7319-7322). In some embodiments, conditionssuitable for cleaving a phosphoramidate linkage can includetrifluoroacetic acid and a temperature range of about 15-45° C., orabout 20-40° C., or about 25-35° C., or about 27-30° C.

In some embodiments, a scissile linkage can include at least onephotolabile internucleosidic linkage. For example, a photolabile linkagecan include an o-nitrobenzyl (Pillai 1987 in “Organic Photochemistry”ed. Padwa N Y; Walker 1988 Journal of Am. Chem. Soc. 110:7170-7177), oran o-nitrobenzyloxymethyl or p-nitrobenzyloxymehtyl group. Otherexamples of photolabile linkages or groups include dimethoxybenzoincarbonates (Pirrung and Bradley 1995 Journal Org. Chem. 60:1116-1117),ortho-nitrophenylethyl-type carbonates and ortho-nitrophenylethyl-typesulfonates (Hasan 1997 Tetrahedron 53:4247-4264; Giegrich 1998Nucleosides and Nucleotides 17:1987-1996; and U.S. Pat. Nos. 5,763,599and 6,153,744). In some embodiments, a photolabile group can be joinedto a 5′ or 3′-hydroxyl group of a nucleoside moiety. In someembodiments, a photolabile linkage can be cleaved with light, includingshort wavelength light such as UV irradiation. For example, aphotolabile linkage can be cleaved with light having a wavelength ofabout 333-550 nm. In some embodiments, conditions suitable for cleavinga scissile linkage can include an ultraviolet photochemical reaction.

In some embodiments, a scissile linkage can include an apurinictetrahydrofuran site. An apurinic tetrahydrofuran site can be cleavablewith an endonuclease, including an endonuclease IV orapurinic/apyrimidinic endonuclease (e.g., AP endo, HAP1, Apex or Ref1).

In some embodiments, a scissile site can include at least one uracilbase. In some embodiments, a uracil base can be cleaved with a uracilDNA glycosylase (UDG) (also referred to as uracil N glycosylase) orformamidopyridine [fapy]-DNA glycosylase. In some embodiments, theexemplary bi-directional sequencing methods can optionally include atemplate strand possessing one or more uracil located along the lengthof the template strand to be cleaved. For example, a first primer can behybridized to the template strand possessing one or more uracil alongits length; the first primer can be extended via template-dependentnucleotide incorporation using a polymerase and dNTPs. The resultingextended primer product/template duplex can undergo UDG digestion tocreate one or more nicks along the template strand. The nicks can betreated (for example, with one or more enzymes such as an endonucleaseand/or an exonuclease) to degrade portions of the template strandcreating one or more single stranded regions within the extended firstprimer product. In some embodiments, the degrading reaction can beperformed or modulated by an enzyme possessing strand displacementactivity and/or 5′-3′ exonuclease activity. Optionally, the singlestranded regions can be extended in the presence of a polymerase anddNTPs to create an extended product (that is substantially complementaryto the first primer product over at least some portion of theirrespective lengths). The disclosed methods can therefore include aprocess for performing bi-directional sequencing.

In some embodiments, the disclosure relates generally to methods forgenerating single-stranded polynucleotides. In some embodiments, thedisclosure provides methods for generating single-stranded nucleicacids. In some embodiments, the method includes hybridizing a firstprimer to a nucleic acid molecule, extending the first primer viapolymerization under polymerization conditions, thereby forming anextended first primer product that is complementary to a portion of thenucleic acid molecule, introducing a nick into a portion of the nucleicacid molecule that is hybridized to the extended first primer product,where the nick includes a free 5′ end and a free 3′ end in the nucleicacid molecule; and degrading the nucleic acid molecule from the free 5′end of the nick using a degrading agent, thereby generatingsubstantially single-stranded extended primer products. In someembodiments, the single-stranded extended primer products can be furtherseparated from the nucleic acid molecules, for example by sodiumhydroxide or heat treatment. In some embodiments, the method includesintroducing a nick in a template strand hybridized to a first extendedprimer product, degrading a portion of the template strand with a stranddisplacement activity or degrading agent, thereby generating asubstantially single stranded polynucleotide, where a portion of theextended first primer product remains hybridized to an undegradedportion of the template strand. Optionally, the method can furtherinclude treating the extended primer product with a separation agent toseparate the extended primer product from the undegraded portion of thetemplate strand. In some embodiments, the separated extended primerproduct can be captured, for example with a binding partner or captureprobe, and sequenced. In some embodiments, the captured extended primerproduct can be hybridized to a second primer and sequenced underpolymerization conditions in the presence of a polymerase and dNTPs. Insome embodiments, the sequencing can include obtaining sequencinginformation from some or all of the nucleotides incorporated during thefirst primer extension and/or the extension of the single-strandedregions within the extended first primer product. In some embodiments,sequencing can occur when a template strand is attached to a supportsuch as, but not limited to a solid support. In some embodiments, thesupport can include a bead, microsphere, nanopore, well, trough, groove,channel, flowcell, microwell, slide or other attachment surface.

In some embodiments, the method includes introducing a nick into thetemplate strand hybridized to the first extended primer product,degrading a portion of the template strand with a degrading agent,thereby generating a substantially single stranded polynucleotide. Insome embodiments, the introducing includes nicking the template strandat one or more sites in the template strand, and then digesting thetemplate strand from at least one nick to form a single-stranded region.

In some embodiments, the introducing includes nicking a nucleic acidmolecule, for example a template strand, and then moving the position ofthe nick using one or more nick translating enzymes. The nicktranslation enzyme can include any enzyme that can move the position ofthe at least one nick to a new position along the nucleic acid strand.In some embodiments, moving the position of the nicks can be catalyzedby one or more enzymes in the presence of a plurality of nucleotides. Insome embodiments, moving the position of the at least one nick caninclude performing a nick translation reaction. In some embodiments, anenzyme that catalyzes nick translation includes an enzyme that couples a5′→3′ polymerization/degradation reaction, or an enzyme that couples a5→3′ polymerization/strand displacement reaction. In some embodiments, anick translation reaction can be catalyzed by any nucleic acidpolymerase having a 5→3′ nucleotide polymerization activity and a 5→3′exonuclease activity. In some embodiments, a nick translation reactioncan be catalyzed by any nucleic acid polymerase lacking a 3→5′exonuclease activity. In some embodiments, a nick translation reactioncan be catalyzed by any DNA polymerase. In some embodiments, a nicktranslation reaction can be catalyzed by any Family A DNA polymerase(also known as pol I family) or any Family B DNA polymerase. In someembodiments, a nick translation reaction can be catalyzed by Klenowfragment. In some embodiments, a nick translation reaction can becatalyzed by E. coli Polymerase I. In some embodiments, a nicktranslation reaction can be catalyzed by one or more thermostableenzymes having 5→3′ nucleotide polymerization activity and a 5→3′exonuclease activity. In some embodiments, a nick translationthermostable enzyme includes Taq polymerase (from Therms aquaticus), Tfipolymerase (from Thermus filiformis), Pfu polymerase (from Pyrococcusfuriosus), Tth (from Thermus thermophilus), Pow polymerase (fromPyrococcus woesei), Tli polymerase (from Thermococcus litoralis), Pol Iand II polymerases (from Pyrococcus abyssi), and Pab (from Pyrococcusabyssi), or a fragment thereof capable of catalyzing the nicktranslation reaction.

In some embodiments, a nick translation reaction can be catalyzed by oneor more enzymes that couples a 5′ to 3′ DNA polymerization and stranddisplacement reaction. In some embodiments, a strand displacingpolymerase includes Taq polymerase, Tfi polymerase, Bst polymerase (fromBacillus stearothermophilus), Tli polymerase, 9° N polymerase, and phi29polymerase, or a fragment thereof capable of catalyzing a DNApolymerization and strand displacement reaction.

In some embodiments, a nick translation reaction can be catalyzed by acombination of a helicase and a DNA polymerase.

In some embodiments, performing any one or more of the hybridizationreaction, nicking reaction, degrading reaction or nick translationreaction can include modulating the reaction. Modulating can optionallyinclude: increasing or decreasing an enzyme concentration; by increasingor decreasing the nucleotide concentration; by increasing or decreasingthe cation concentration; by increasing or decreasing a reactiontemperature, reaction time and/or pH, and the like. The modulating caninclude increasing or decreasing the rate of the reaction, increasing ordecreasing the yield of product of the reaction, and the like.

In some embodiments, the method further includes degrading at least onenucleic acid molecule at any point in time during the sequencingprocess. As used herein, the term “degrading” and its variants refer toany process whereby the physical integrity of at least some portion of anucleic acid strand is disrupted sufficiently that a polymerase cannotprocess along that portion of the nucleic acid strand. In someembodiments, “degrading” can include treating the nucleic acid moleculeto be degraded with a degrading agent. In some embodiments “degrading”can include disruption of the 5′-3′ phosphodiester bonds between any twoor more contiguous nucleotides within the region that is degraded. Insome embodiments, “degrading” includes processes whereby thephosphodiester bonds remain intact while the base pairing interactionsof any two or more contiguous nucleotides within the degraded regionwith corresponding residues in another nucleic acid strand aredisrupted, or the physical integrity of the degraded region is otherwiseundermined. In some embodiments, the degrading agent can besite-specific to a site within the nucleic acid; for example, thedegrading agent may cleave the nucleic acid molecule in a site-specificmanner. In some embodiments, the nucleic acid molecule to be degraded,which can include the template molecule, an extended primer product, orboth, can include a site that is selectively recognized by the degradingagent; optionally, the site can also be cleaved or otherwise degraded bythe degrading agent. In some embodiments, the degrading agent caninclude a restriction enzyme, and the nucleic acid molecule to bedegraded can include a restriction recognition site. In someembodiments, the degrading can include degrading a portion of at leastone strand within a nucleic acid duplex. In some embodiments, thedegrading includes degrading a portion of at least one strand of duplexafter the strand has been nicked. The degrading can include using adegrading agent to disrupt the integrity of the nucleic acid strand. Thedegrading agent can optionally bind to the 5′ end of a nick within thenucleic acid strand and degrade the nucleic acid strand in the 5′ to 3′direction. Alternatively, the degrading agent can optionally bind to the3′ end of a nick within the nucleic acid strand and degrade the nucleicacid strand in the 3′ to 5′ direction. In some embodiments, a degradingagent is added to a nucleic acid duplex formed via hybridization of theextended first primer product and the nucleic acid template. In someembodiments, the degrading agent degrades one or more free 5′endspresent within the nucleic acid duplex.

In some embodiments, the degrading agent is an enzyme that is capable ofdegrading a nucleic acid molecule via exonucleolytic digestion, eitherin the 3′ to 5′ direction, or the 5′ to 3′ direction, or both. Forexample, the enzyme can be Exonuclease I or Exonuclease III, whichpossess 3′ to 5′ exonuclease activity. For example, the enzyme can be T5or T7 exonuclease, which possesses 5′ to 3′ exonuclease activity. In atypical embodiment, a duplex including the template strand hybridized tothe first extended primer product is nicked within the template strandand contacted with a 5′ to 3′ exonuclease (e.g., T5 or T7 exonuclease),which binds to the 5′ end of the nick within the template strand anddegrades a portion of the template strand located 3′ of the nick, wherethe degradation occurs in the 5′ to 3′ direction.

In some embodiments, the method includes degrading at least a portion ofthe template strand prior to extending the second primer. In someembodiments, the degrading is performed before or after hybridizing thesecond primer to the extended first primer product. In some embodiments,the degradation reaction can be modulated by: increasing or decreasingan enzyme concentration; by increasing or decreasing the cationconcentration; by increasing or decreasing a reaction temperature,reaction time and/or pH, and the like.

In some embodiments, the method includes displacing at least a portionof the template strand prior to extending the second primer. In someembodiments, the displacing is performed before or after hybridizing thesecond primer to the extended first primer product. In some embodiments,the displacing reaction can be performed or modulated by an enzymepossessing strand displacement activity and/or 5′-3′ exonucleaseactivity.

In some embodiments, a primer can be treated with chain-terminatingnucleotide such as ddATP, ddGTP, ddCTP or ddTTP to prevent continuedextension of the primer. In this example, the addition of achain-terminating nucleotide which lacks a 3′-OH group required for theformation of a phosphodiester bond with an adjacent nucleotide, caninhibit further forward primer extension. In some embodiments, theprimer can be treated with apyrase or phosphatase after the polymerasefill-in or primer extension step. In some embodiments, the primer issubstantially resistant to degradation, or includes a nucleic acidsequence that is substantially resistant to degradation. For example, aprimer can include at least one locked nucleic acid (LNA). A lockednucleic acid can be resistant to cleavage or degradation by a degradingagent (e.g., exonuclease). In another example, a primer can include atleast one phosphorothioate linkage. A phosphorothioate linkage can beresistant to exonuclease cleavage (e.g., exonuclease III). In anotherexample, a primer can include at least one nuclease-resistant linkage.In some embodiments, a nuclease-resistant primer can be hybridized tothe template strand.

Generally, the nucleic acid obtained by extension of a first primerand/or a second primer can be sequenced using one or more enzymes. In atypical embodiment, the enzyme is a polymerase. In some embodiments, thesequencing reaction can be modulated by: increasing or decreasing anenzyme concentration; by increasing or decreasing the nucleotideconcentration; by increasing or decreasing the cation concentration; byincreasing or decreasing a reaction temperature, reaction time and/orpH, and the like. In some embodiments, sequencing reactions can beperformed in the presence of appropriate buffers. In some embodiments, asequencing buffer can optionally include a detergent and/or an additive.For example, the buffer can include one or more detergents such as, butnot limited to, TWEEN-20, SDS, TRITON, and the like. In someembodiments, the sequencing buffer can include an additive such as, butnot limited to, polyvinylpyrrolidone (e.g., povidone or crospovidone),poly(4-vinylphenol), and vinylpyrrolidone/vinyl acetate copolymer (e.g.,copovidone). In some embodiments, the additive can include two or moremonomers of N-vinyl-pyrrolidone, including modified polymers thereof.Modified polymers of poly(N-vinyl-pyrrolidone) comprisemonofunctionalized (e.g., hydroxyl or carboxy end group), side-chainconjugates (e.g., poly- and multifunctional side chains), and graftedcopolymers. In some embodiments, polyvinylpyrrolidone includes variousmolecular weight polymers including average molecular weights of about 5kD-55 kD, for example 10 kD, 29 kD, 40 kD, and 55 kD molecular weightcompounds. In some embodiments, an additive (e.g., polyvinylpyrrolidone(PVP)) can be present in the buffer at about 0.1-8%, or about 1-2%, orabout 2-3%, or about 3-4%, or about 4-5%, or about 5-6%, or about 6-7%,or about 7-8%. In some embodiments, a sequencing reaction can beconducted in the presence of one or more reducing agents such asdithiothreitol (DTT) or tris(2-carboxyethyl)phosphine (TCEP).

In some embodiments, the disclosure generally relates to methods forobtaining sequence information from a nucleic acid template linked to asupport, including hybridizing a first primer to a template strandlinked to a support, sequencing a portion of the nucleic acid templateby synthesis, where the sequencing by synthesis includes extending thefirst primer via template-dependent nucleic acid synthesis, therebyforming an extended first primer product that is complementary to aportion of the nucleic acid template. In some embodiments, the methodfurther includes introducing a nick into a portion of the templatestrand that is hybridized to the extended first primer product, wherethe nick includes a free 5′ end and a free 3′ end in the templatestrand, degrading a portion of the template strand from the free 5′ endof the nick using a degrading agent, where a portion of the extendedfirst primer remains hybridized to an undegraded portion of the templatestrand, and sequencing at least some of the single-stranded portion ofthe extended first primer by synthesis. In some embodiments,substantially all of the extended first primer product is sequenced. Insome embodiments, substantially all of the single-stranded portion ofthe extended first primer is sequenced. In some embodiments, the firstprimer provides sequence information in a forward or first direction. Insome embodiments, sequencing of the single-stranded portion of theextended first primer product provides sequencing information in areverse or second direction. In some embodiments, the method provides aprocess by which to obtain bi-directional sequencing information from atemplate strand. In some embodiments, bi-directional sequencing canimprove sequencing throughput and/or sequencing accuracy as compared toa single end sequencing reaction (e.g., a forward or reverse sequencingreaction). In some embodiments, bi-directional sequencing includeslabel-free or ion based sequencing. In some embodiments, bi-directionalsequencing includes optically detectable or fluorescence basedsequencing. In some embodiments, the template strand can be linked tothe support through the 5′ end of the template strand. In someembodiments, the template strand can be linked to the support through atleast one nucleotide in the template strand that can be situated 5′ ofthe nick site in the template. In some embodiments, the first primer isresistant to degradation by the degrading agent. In some embodiments,the extended first primer product is resistant to degradation by thedegrading agent. In some embodiments, the degrading agent can include a5′-3′ exonuclease, and the degrading can further include digesting thetemplate strand from the free 5′ end of the nick using the 5′-3′exonuclease. In some embodiments, sequencing of at least some portion ofthe single-stranded portion of the extended first primer product caninclude extending the free 3′ end of the nick via nucleic acidsynthesis, thereby synthesizing a nucleic acid molecule that iscomplementary to at least some portion of the single-stranded portion ofthe extended first primer. In some embodiments, sequencing at least someof the single-stranded portion of the extended first primer product caninclude hybridizing a second or reverse primer to a sequence within thesingle-stranded portion of the extended first primer product, andextending the second or reverse primer using a polymerase. In someembodiments, the polymerase includes a thermostable DNA polymerase.

In some embodiments, the degrading agent can initiate degradation at thefree 3′ end of a nick. In some embodiments, the degrading agent cancatalyze nucleic acid degradation (e.g., exonuclease activity) coupledwith nucleotide polymerization. In some embodiments, the degrading agentcan catalyze degrading coupled with nucleotide polymerization in a 5′ to3′ direction. In some embodiments, the degrading agent can catalyzetemplate-dependent nucleotide polymerization. In some embodiments, thedegrading agent can generate a nucleic acid molecule that is at leastpartially complementary to the extended first primer. In someembodiments, the degrading agent includes a polymerase. In someembodiments, the order of polymerized nucleotides (e.g., catalyzed bythe polymerase) can be monitored to determine the nucleotide sequence ofthe nucleic acid molecule. In some embodiments, the sequencing cancomprise sequencing at least some of the single-stranded portion of theextended first primer. In some embodiments, the sequencing can comprisesequencing at least some of the single-stranded portion of the extendedfirst primer product and can further include hybridizing a reverseprimer to a sequence within the single-stranded portion of the extendedfirst primer product, and extending the reverse primer using apolymerase.

In some embodiments, methods for nucleic acid sequencing includehybridizing a first primer to a distal end of a nucleic acid strandhaving a distal and proximal end, where the proximal end of the nucleicacid strand is linked to a solid support, extending the hybridized firstprimer in the direction of the proximal end of the nucleic acid strandand the solid support, thereby forming an extended first primer productthat is complementary to a portion of the nucleic acid strand andobtaining a first sequencing read. In some embodiments, the method canfurther include introducing a site-specific nick into the proximal endof the nucleic strand hybridized to the extended first primer product,degrading a portion of the nucleic acid strand, thereby generating asingle-stranded portion within the extended first primer product, wherea portion of the extended first primer product remains hybridized to thenucleic acid strand and extending the single-stranded portion within theextended first primer product, thereby obtaining a second sequencingread. In some embodiments, extending is performed via template-dependentnucleic acid synthesis. In some embodiments, the first primer or theextended first primer product is nuclease resistant. In someembodiments, the solid support includes an Ion Sphere Particle (ISP). Insome embodiments, the nucleic acid sequencing includes bi-directionalsequencing. In some embodiments, the sequencing includes a first orforward sequencing read coupled with a second or reverse sequencingread. In some embodiments, the sequencing includes obtaining sequencinginformation that is reversed relative to sequencing information obtainedin an anti-parallel orientation. In some embodiments, the nucleic acidsequencing is label-free or ion based sequencing. In some embodiments,introducing a site-specific nick is performed by an enzyme. In someembodiments, the site-specific nicking enzyme is a restriction enzyme.In some embodiments, extending the first primer or second primer isperformed by a DNA polymerase. In some embodiments, the nucleic acid tobe sequenced is a DNA, cDNA, RNA, mRNA, or DNA/RNA hybrid. In someembodiments, the nucleic acids to be sequenced are obtained from alaboratory, a morgue, a clinical specimen (e.g., FFPE or biopsies), aDNA database, a patient (e.g., a hair, blood or saliva sample), a livingorganism, or from the circulatory system of mammals (e.g., as cell-freecirculating DNA).

In some embodiments, the methods for nucleic acid sequencing can furthercomprise consolidating the first and second sequencing reads (e.g. FIG.4). In some embodiments, consolidating can comprise aligning the firstand second sequencing reads against a reference sequence. In someembodiments, aligning the first and second sequencing reads against areference sequence can determine the presence of deletions, insertions,variations, inversions, translocations, mutations or mismatches in thenucleic acid strand as compared to the reference sequence. In someembodiments, paired-end sequencing methods according to the disclosurecan detect splice variants and fusion transcripts. In some embodiments,aligning the first and second sequencing read can identify the nature ofthe mutations, mismatches, insertions, deletions or variations in thenucleic acid strand.

For example, in some embodiments the disclosure relates generally to amethod for obtaining sequence information from a nucleic acid templatelinked to a support in both directions, comprising: hybridizing a firstprimer including to a template strand linked to a support. The templatestrand can include a nicking site, for example a site specific nickingsite. The first primer can then be used to prime extension in the“forward” direction, thereby sequencing a portion of the nucleic acidtemplate by synthesis in the “forward” direction. The sequencing caninclude extending the first primer via template-dependent nucleic acidsynthesis using a polymerase, thereby forming an extended first primerproduct that is complementary to a portion of the nucleic acid template.The extending optionally proceeds past nicking site in the template,such that the extended first primer product includes sequence that iscomplementary to the nicking site in the template strand. The extendedfirst primer product can be hybridized to the nucleic acid template toform a first nucleic acid duplex, of which the template strand is linkedto the support.

In some embodiments, the template nucleic acid strand can be an isolatedDNA nucleic acid molecule. In some embodiments the template nucleic acidstrand can include a nucleic acid molecule prepared from emulsion PCR orbridge PCR. In some embodiments, the template nucleic acid strand can beenzymatically prepared from high molecular weight DNA, for example usingan Ion Xpress Plus Fragment Library Kit (Life Technologies, Part No.4468987). In some embodiments, the template nucleic acid strand can beprepared from sheared DNA such as mechanically sheared DNA or chemicallytreated DNA such as formalin-fixed paraffin-embedded (FFPE) DNA. In someembodiments the template nucleic acid strand includes an insert lengthof between about 100 and about 500 base pairs. In some embodiments thetemplate nucleic acid strand includes an insert length of greater than500 base pairs, greater than 600 base pairs, or more. In someembodiments, the length of the template strand coupled withbi-directional sequencing allows for highly precise alignment of DNAreads of high quality (>1 gigabyte of data at AQ17). In someembodiments, the length of the template strand coupled with bi-directionsequencing allows for highly precise alignment of DNA reads of highquality (>1 gigabyte of data at AQ20) in both the forward and/or reverseread (e.g., Example 22). In some embodiments, the first primer isreferred to as the “forward” primer and the extended first primerproduct (EFPP) is referred to as the extended forward primer product.The sequencing can optionally include detecting a byproduct of at leastone nucleotide incorporation. In some embodiments, detecting a byproductof at least one nucleotide incorporation can be achieved using a fieldeffect transistor (FET). In some embodiments, detecting a byproduct ofat least one nucleotide incorporation can occur using an ion-sensitivefield effect transistor (ISFET). In some embodiments, the byproduct ofnucleotide incorporation can include a hydrogen ion, an inorganicpyrophosphate or an inorganic phosphate. In some embodiments, thesequencing can optionally include detecting the incorporation of anoptically labeled nucleotide.

In some embodiments, the method further includes introducing a nick intothe first nucleic acid duplex. Typically, the nick is introduced into aportion of the template strand that is hybridized to the extended firstprimer product. In some embodiments, introducing a nick into the firstnucleic acid duplex can include nicking the nicking site in the templatestrand using a suitable nicking agent. For example, the nicking site inthe template strand can be a site specific nicking site, and the nickingagent can be a nicking enzyme capable of nicking the site specificnicking site. Typically, the nick includes a free 5′ end and a free 3′end.

In some embodiments, the method further includes degrading a portion ofthe template strand. The degrading agent can include a 5′-3′exonuclease, and the degrading further includes digesting the templatestrand from the free 5′ end of the nick using the 5′-3′ exonuclease. Ina typical embodiment, the degrading includes degrading the templatestrand from the free 5′ end of the nick in the 5′ to 3′ direction usinga suitable degrading agent (e.g., a 5′ to 3′ exonuclease), while leavingintact or undegraded the 3′ end of the nick and any nucleotides that arecovalently linked to the free 3′ end of the nick, either directly orthrough other nucleotides (such intact portion including the 3′ end ofthe nick and any nucleotides covalently linked thereto being referred toas the “residual portion” of the template strand). In some embodiments,the degrading generates a single-stranded portion of the extended firstprimer product, wherein the residual portion of the template strandremains hybridized to a region within the extended first primer product.In some embodiments, a removal or wash step is carried out prior toextension of the single-strand portion of the template strand but afterextension of the extended first primer product.

In some embodiments, the method further includes sequencing at leastsome of the single-stranded portion of the extended first primerproduct. Such sequencing can include extending the free 3′ end of thenick via nucleic acid synthesis, thereby synthesizing a nucleic acidmolecule that is complementary to at least some of the single-strandedportion of the extended first primer. For example, in a typicalembodiment the residual portion of the template strand can be used toprime extension in the “reverse” direction, thereby sequencing a portionof the nucleic acid template by synthesis in the “reverse” direction.The sequencing by synthesis can include extending the residual portionof the template strand via template-dependent nucleic acid synthesisusing a polymerase, thereby obtaining a “reverse” read of the templatestrand. Alternatively, in some embodiments sequencing at least some ofthe single-stranded portion of the extended first primer product caninclude use of a separate primer. For example, such sequencing caninclude hybridizing a reverse primer to a sequence within thesingle-stranded portion of the extended first primer, and extending thereverse primer using a polymerase. The sequencing can optionally includedetecting a byproduct of at least one nucleotide incorporation using afield effect transistor (FET). In some embodiments, the method furthercomprises obtaining both a first (forward) and a second (reverse)sequencing read. In some embodiments, the method further comprisesobtaining both a sense sequencing read and an antisense sequencing read.In some embodiments, the method includes sequencing in a firstorientation, optionally followed by sequencing in an orientation that isreversed relative to the first sequencing read. In some embodiments, thefirst and second sequencing reads are aligned against a referencesample. In some embodiments, the first and second sequencing reads arealigned against a reference sample to determine the presence of variantsin the first or second sequencing reads as compared to the referencesample. In some embodiments, the first and second reads are aligned toprovide a de novo nucleic acid sequence. In some embodiments, the firstand/or second sequencing reads can be used to determine the presence ofone or more insertions, deletions, mismatches or other nucleotide errorsin the first or second sequencing reads when compared against areference sample. In some embodiments, the first and second sequencingreads are consolidated. In some embodiments, the consolidation processimproves sequencing accuracy when compared to a non-consolidatedsequencing read. In some embodiments, the consolidation process improvessequencing accuracy when compared to a single end sequencing read.

In some embodiments, the template strand is linked to the supportthrough the 5′ end of the template strand. In some embodiments, thetemplate strand is linked to the support through at least one nucleotidein the template that is situated 5′ of the nick site in the template.

In some embodiments, the first primer is resistant to degradation by thedegrading agent. For example, where the degrading agent is T7exonuclease, the first primer can include one or more nucleotides thatare resistant to digestion by T7 exonuclease. In another example, wherethe degrading agent is T5 exonuclease, the first primer can include oneor more nucleotides that are resistant to digestion by T5 exonuclease.

In some embodiments, additional components may be added to thebi-directional sequencing method such as, but not limited to, cations,salts, polypeptides, polymers, detergents, surfactants and excipients tooptimize one or more of the steps. In some embodiments, additionalcomponents such as, but not limited to, cations, salts, polypeptides,polymers, detergents, surfactants and excipients can be added to one ormore of the degrading, nicking, extension or sequencing steps.

In some embodiments, the sequencing and/or extension reactions can beoptimized by one of ordinary skill in the art to achieve the desiredsequencing information such as sequencing accuracy, yield, totalthroughput and/or nucleotide sequence information. In some embodiments,the sequencing throughput achieved in a bi-directional sequencingreaction (i.e., totality of forward and reverse reads) can exceed 1, 2,3, or 4 gigabytes of data at AQ20. In some embodiments, the sequencingthroughput achieved with a bi-directional sequencing reaction can exceed1, 2, 3, or 4 gigabytes of data or more at AQ17.

In some embodiments, the disclosure relates generally to systems forsequencing nucleic acids, comprising: template nucleic acids, one ormore primers, one or more polymerases, one or more degrading agents, anddeoxyribonucleotide triphosphates. In some embodiments, the disclosuregenerally relates to systems for sequencing nucleic acids in a first(forward) and second (reverse) orientation. In some embodiments, thedisclosure generally relates to systems for sequencing nucleic acids ina bi-directional orientation. In some embodiments, systems forsequencing nucleic acids further comprise one or more nicking enzymes.In some embodiments, systems for sequencing nucleic acids furthercomprise any combination of: buffers; cations; solid-supports; one ormore nick translation enzymes, reagents for nucleic acid purification;reagents for nucleic acid amplification; endonuclease(s); kinase(s);phosphatase(s); and/or nuclease(s).

Provided herein are compositions for immobilizing a substantiallycomplementary sequence such as, but not limited to, a sequencing primerin an orientation that is reversed compared to the orientation of thetemplate strand.

Provided herein are primers comprising at least one functional sequenceor site in any combination and in any order, including: a cleavageresistant site, a priming sequence, a cross-linking sequence, arestriction endonuclease recognition sequence, and/or a nickingendonuclease recognition sequence. The functional sequence or sitepermits nucleic acid manipulations, such as cleavage, primer extension,digestion, strand displacement or cross-linking. In some embodiments,the primers can function as a catalyst for primer extension reactions.

In some embodiments, the primers can be hybridized to the templatestrand. The various functional sequences or sites on the primers permitvarious nucleic acid manipulations that can be used to generate extendedprimer products that include a sequence substantially complementary thetemplate strand with an orientation that is reversed compared to thetemplate strand.

In some embodiments, the disclosure relates generally to kits forsequencing nucleic acids. In some embodiments, the disclosure relatesgenerally to kits for sequencing nucleic acids in a first (forward) andsecond (reverse) orientation. In some embodiments, the disclosurerelates generally to kits for improving sequencing accuracy. In someembodiments, the disclosure relates generally to kits for generatingsingle-stranded nucleic acids. In some embodiments, the kits include anyreagent that can be used to conduct nucleic acid sequencing. In someembodiments, kits for sequencing include any reagent that can be used toconduct nucleic acid sequencing in a bi-directional method.

In some embodiments, the disclosure relates generally to kits comprisinga primer having an exonuclease resistant nucleotide sequencesubstantially complementary to the template nucleic acid to besequenced, a polymerase, dNTPs, a nicking enzyme and a degrading enzyme.In some embodiments, the kit can further include a support and/or one ormore primers or adaptors that can be used to link the template nucleicacid to the support. In some embodiments, the exonuclease resistantnucleotide sequence can include one, two, three, four, five or morephosphorothioate residues. In some embodiments, the kit can furtherinclude a support such as a bead, particle, microparticle, slide, array,and the like.

In some embodiments, the kits include any combination of: buffers;cations; one or more primers; one or more enzymes; one or more degradingagent(s); one or more nucleic acid nicking enzyme(s); one or more nicktranslation enzyme(s); one or more nucleotides; one or moredeoxyribonucleotide triphosphates; reagents for nucleic acidpurification; and/or reagents for nucleic acid amplification. In someembodiments, the kits include any combination of: endonuclease(s);exonuclease(s); polymerase(s); ligase(s); kinase(s); phosphatase(s);and/or nuclease(s).

Embodiments of the present teachings can be further understood in lightof the following examples, which should not be construed as limiting thescope of the present teachings in any way. Although the presentdescription described in detail certain exemplary embodiments, otherembodiments are also possible and within the scope of the presentinvention. Variations and modifications will be apparent to thoseskilled in the art from consideration of the specification and figuresand practice of the teachings described in the specification andfigures, and the claims.

EXAMPLES Example 1

FIG. 1 depicts an exemplary embodiment according to the methods of thedisclosure involving paired-end sequencing using an ion-based sequencingsystem. A nucleic acid template strand, which includes a site specificnicking site at or near the 5′ end, is linked to a solid support (here,an Ion Sphere™ particle) through the 5′ end of the template strand.

A “forward” sequencing primer (A Seq Primer) is hybridized to the 3′ endof the template strand, and the hybridized primer:template system isplaced within an Ion Chip, where the hybridized: template system isdeposited in a microwell within the Ion Chip, which is then placedwithin the Ion Torrent PGM™ sequencing system (Life Technologies, CA).Nucleotides are then flowed serially into the Ion Chip, and the forwardprimer is extended via stepwise template-dependent nucleic acidincorporation using a polymerase to form an extended forward primerproduct. As each nucleotide is incorporated in a stepwise fashion intothe extending forward primer, such incorporation is detected via the FETlinked to the microwell in the Ion Chip, providing a “forward”sequencing read of the template strand. The forward primer is extendedpast the site specific nicking site in the template strand, and theresulting extended forward primer product therefore includes sequencecomplementary to the site-specific nicking site.

After the forward primer extension is completed (and the forwardsequencing read is obtained), the Ion Chip is flushed with a solutionincluding a nicking agent (here, a nicking enzyme that can cleavespecifically at the site-specific nicking site within the templatestrand). Conditions are adjusted to facilitate nicking of the templatestrand at the site-specific nicking site in the template strand usingthe nicking agent (shown as site-specific exonickase).

After nicking is completed, the Ion Chip is flushed with a degradingagent (here, T7 exonuclease) that is capable of digesting the templatestrand from the 5′ end of the nick in the 5′ to 3′ direction, leavingthe 3′ end of the nick and associated upstream template sequence intactwhile simultaneously creating a single-stranded region within theextended first primer product (the intact portion hereafter beingreferred to as the “residual portion” of the template strand). Theresidual portion remains hybridized to the complementary sequence in theextended forward primer product and also remains linked to the IonSphere™ particle at its 5′ end. The residual portion of the templatestrand is then used to prime extension in the “reverse” direction toprovide a “reverse” sequencing read. To sequence in the “reverse”direction, each of the four nucleotide types (A, C, G and T) are flushedserially into the Ion Chip, and any consequent nucleotide incorporationis detected using the FET that is operationally associated with themicrowell including the Ion Sphere™ particle and associated nucleic acidmolecule.

Typically, an exchange of reagents into the Ion Chip is preceded by awash step to remove prior reagents (enzymes, nucleotides, etc) so thatthe succeeding reaction is not contaminated by reagents from priorsteps.

Example 2

FIG. 2 depicts an exemplary embodiment according to methods of thedisclosure involving paired-end sequencing using an ion-based sequencingsystem. A nucleic acid template strand (T), which includes a sitespecific nicking site at or near the 5′ end, is linked to a solidsupport (here, an Ion Sphere™ particle) through the 5′ end of thetemplate strand.

A “forward” sequencing primer (green primer (A)) is hybridized to the 3′end of the template strand (yellow primer complement (A′)), and thehybridized primer:template system is placed within an Ion Chip, wherethe hybridized: template system is deposited in a microwell in the IonChip, which is placed within an Ion Torrent PGM™ sequencing system. Asequencing polymerase (P) is bound to the hybridized: template system.dNTPs are then flowed serially into the Ion Chip, and the forward primeris extended via stepwise template-dependent nucleic acid incorporationusing a sequencing polymerase to form an extended forward primer product(T′). As each nucleotide is incorporated in a stepwise fashion into theextending forward primer, such incorporation is detected via the FETlinked to the microwell in the Ion Chip, providing a “forward”sequencing read of the template strand.

After the forward primer extension is completed (and the forwardsequencing read is obtained), the Ion Chip is flushed with a solutionincluding a nicking agent (here, a nicking enzyme that can cleavespecifically at a site-specific nicking site within the templatestrand). Conditions are adjusted to facilitate nicking of the templatestrand at the site-specific nicking site in the template strand usingthe nicking agent.

After nicking is completed, the Ion Chip is flushed with a degradingagent that is capable of digesting the template strand from the 5′ endof the nick in the 5′ to 3′ direction, leaving the 3′ end of the nickand associated upstream template sequence intact (the intact portionhereafter being referred to as the “residual portion” of the templatestrand (Ion-Sphere Bound Oligonucleotide)). The residual portion remainshybridized to the complementary sequence in the extended forward primerproduct and also remains linked to the Ion Sphere™ particle at its 5′end. The residual portion is then used to prime extension in the“reverse” direction to provide a “reverse” sequencing read. To sequencein the “reverse” direction, each of the four nucleotide types (A, C, Gand T) are flushed serially into the Ion Chip, and any consequentnucleotide incorporation is detected using the FET that is operationallyassociated with the microwell including the Ion Sphere™ particle andassociated nucleic acid. Typically, an exchange of reagents into the IonChip is preceded by a wash step to remove prior reagents (enzymes,nucleotides, etc) so that the succeeding reaction is not contaminated byreagents from prior steps.

Example 3

FIG. 3 depicts an exemplary embodiment according to the methods of thedisclosure involving paired-end sequencing using an ion-based sequencingsystem. A nucleic acid template strand (T), which includes a sitespecific nicking site at or near the 5′ end, is linked to a solidsupport (here, an Ion Sphere™ particle) through the 5′ end of thetemplate strand.

A “forward” sequencing primer (green primer (A)) is hybridized to the 3′end of the template strand (yellow primer complement (A′)), and thehybridized primer:template system is deposited in a microwell within anIon Chip, which is placed within an Ion Torrent PGM™ sequencing system.A sequencing polymerase (P) is bound to the hybridized: template system.dNTPs are then flowed serially into the Ion Chip, and the forward primeris extended via stepwise template-dependent nucleic acid incorporationusing a sequencing polymerase to form an extended forward primer product(T′). As such, the extended forward primer product is sequenced from thedistal end. As each nucleotide is incorporated in a stepwise fashioninto the extending forward primer, such incorporation is detected viathe FET linked to the microwell in the Ion Chip, providing a “forward”sequencing read of the template strand. The forward primer is extendedpast the site specific nicking site in the template strand (denoted by acarot), and the resulting extended forward primer product thereforeincludes sequence complementary to the site-specific nicking site.

After the forward primer extension is completed (and the forwardsequencing read is obtained), the Ion Chip is flushed with a solutionthat removes the sequencing polymerase from the template and/or extendedforward primer product. Conditions are adjusted to facilitatedegradation of the template strand by applying a degrading agent(denoted as dark circle) to the Ion Chip. The degrading agent digeststhe template strand from the 5′ end of the nick in the 5′ to 3′direction, leaving the 3′ end of the nick and associated upstreamsequence intact (the intact portion hereafter being referred to as the“residual portion” of the template strand). The 3′ sequencing primerattached to the extended primer product (A) (and optionally theion-sphere bound oligonucleotides B and B′) are resistant to the actionof the degrading agent, i.e., are nuclease resistant. The residualportion of the template strand remains hybridized to the complementarysequence in the extended forward primer product and also remains linkedto the Ion Sphere™ particle at its 5′ end. A sequencing polymerase (P)is then added to the Ion Chip and the residual portion is then used toprime extension in the “reverse” direction to provide a “reverse”sequencing read. To sequence in the “reverse” direction, each of thefour nucleotide types (A, C, G and T) are flushed serially into the IonChip, and any consequent nucleotide incorporation is detected using theFET that is operationally associated with the microwell including theIon Sphere™ particle and associated nucleic acid. Typically, an exchangeof reagents into the Ion Chip is preceded by a wash step to remove priorreagents (enzymes, nucleotides, etc) so that the succeeding reaction isnot contaminated by reagents from prior steps. In this exemplaryembodiment, the reverse read is sequenced from the proximal end.

Example 4

A paired-end library was made using the Ion Fragment Library Kit (LifeTechnologies, Part No. 4466464), hereby incorporated by reference in itsentirety, essentially according to the protocols provided in the IonXpress™ Fragment Library Kit User Guide (Life Technologies, Part No.4468987), hereby incorporated by reference in its entirety with thefollowing modifications. E. coli genomic DNA was enzymatically digestedusing Ion Shear™ Reagents Kit (Life Technologies, Part No. 4468655) for15 minutes at 37° C. to obtain a DNA fragment distribution between 75and 200 bases. DNA fragments were then purified using Agencourt® AMPure®XP Reagent and ligated using DNA ligase at room temperature for 30minutes to paired-end specific Ion Adapters (sold as a component of theIon Fragment Library Kit (Life Technologies, Part No. 4466464),essentially according to the protocols provided in the Ion Xpress™Fragment Library User Guide), which contain a Nt.BbvCI nick site andligation to an Ion Torrent PGM™ key sequence on an alternative P1 primer(5-CCTCTCTATGGGCAGTCGGTGATCCTCAGC-3 (SEQ ID NO: 1)). Size selection for180 bp mean library size was performed on a Pippin Prep™ with 2% agarosecassette (Sage Science CSD-2010), essentially according to themanufacturer's instructions. The size selected DNA library was thenpurified, nick translated and amplified in a thermocycler for 7 cyclesessentially according to the protocol of the Ion Xpress™ FragmentLibrary Kit User Guide. The amplified DNA was purified using Agencourt®AMPure® XP Reagent and the DNA was eluted from the beads to a new 1.5 mlLoBind Tube (Eppendorf).

An aliquot of the eluted DNA library sample was analyzed using theAgilent Technologies 2100 Bioanalyzer™ to ensure the library was of theexpected size distribution. The library was quantitated to determine thelibrary dilution that results in a concentration within the optimizedtarget range for Template Preparation (e.g., PCR-mediated addition oflibrary molecules onto Ion Sphere™ Particles). The DNA library istypically quantitated using an Ion Library Quantitation Kit (qPCR) (LifeTechnologies, Part No. 4468802) or Bioanalyzer™ (Agilent Technologies,Agilent 2100 Bioanalyzer) to determine the molar concentration of thelibrary, from which the Template Dilution Factor is calculated. Forexample, instructions to determine the Template Dilution Factor byquantitative real-time PCR (qPCR) can be found in the Ion LibraryQuantitation Kit User Guide (Life Technologies, Part No. 4468986),hereby incorporated by reference in its entirety.

After quantification of the DNA library yield, the library was clonallyamplified onto Ion Sphere™ Particles (ISPs) using the Ion Xpress™Template Kit (Life Technologies, Part No. 4469001), hereby incorporatedby reference in its entirety, essentially according to the protocols inthe Ion Xpress™ Template Kit User Guide v2.0 (Life Technologies, PartNo. 4469004), hereby incorporated by reference in its entirety, with thefollowing exceptions. Double the input of recommended ISPs was used toincrease overall yield. Enrichment for template positive ISPs wasperformed on an Ion OneTouch™ ES System (Life Technologies, Part No.4467889) essentially according to the protocols of the Ion OneTouch™Template Kit User Guide (Life Technologies, Part No. 4468660), herebyincorporated by reference in the their entireties. After completing thetemplate positive enrichment step, 5 ul of 100 uM Seq Primer A(5-C*C*A*T*C*T*CATCCCTGCGTGTCTCCGAC-3, where *=phosphorothioate bond(SEQ ID NO: 2) was hybridized to 10 million template positive ISPs. Thehybridized template positive ISPs where then sequenced on a Ion TorrentPGM™ sequencer (Life Technologies, Part No. 4462917), essentiallyaccording to the protocols provided in the Ion Sequencing Kit User Guidev2.0 (Life Technologies, Part No. 4468997), hereby incorporated byreference in its entirety, using Ion PGM™ Supplies Kit (LifeTechnologies, Part No. 4468996), Ion Sequencing Reagents Kit (LifeTechnologies, Part No. 4468995) and Ion PGM™ Reagents Kit (LifeTechnologies, Part No, 4468994). In this example, the template positiveISPs were applied to an Ion Torrent 314™ Chip (Life Technologies, PartNo. 4462923) for sequencing.

Following initialization of the PGM sequencer and calibration, a firstrun (forward read) was performed on the 314™ chip. The 314™ experimentalchip was removed from the Ion Torrent PGM™ sequencer and a dummy chipwas put on the pariposer with the squid clamp shut. The insertion of thedummy chip allowed the PGM™ sequencer to maintain functionality betweenthe two runs performed in this example, without resetting the overallsystem or run parameters.

100 ul of Enzyme Denaturation Solution (EDS) containing TE pH 8.0 (SigmaT9285), 2% SDS (Sigma L4522), and 50 mM NaCl (Sigma S-3014) wasdispensed into the experimental 314™ chip, incubated for 1 minute atroom temperate, and then removed from the 314™ chip flow cell. Threewashes of 100 ul EDS followed. Next, the 314™ experimental chip waswashed three times with 100 ul 1× Thermopol Buffer (New England BioLabs,Part No. B9004S). After the final wash, the remaining buffer was removedfrom the 314™ flow cell. 5 ul of Fill-in Solution (containing: 2 ulSequencing Polymerase (sold as a component of the Ion SequencingReagents Kit, Life Technologies, Part No. 4468995), 4 ul dNTPs (1 ul ofeach dNTP sold as a component of the Ion Sequencing Reagents Kit, LifeTechnologies, Part No. 4468995), 2 ul Thermopol Buffer (New EnglandBioLabs, Part No. B9004S) and 12 ul nuclease-free water) was added tothe 314™ experimental chip and incubated at room temperature for 10minutes with size 4 rubber gaskets covering both ports. The use ofrubber gaskets was to ensure the solution did not evaporate. Afterincubation, 100 ul of EDS was dispensed into the 314™ experimental chip,incubated for 1 minute at room temperature and then removed from the314™ flow cell. Three washes of 100 ul EDS followed. Next, the chip waswashed three times with 100 ul 1× Buffer 4 (New England BioLabs, PartNo. B7004S). After the final wash, the remaining buffer was removed fromthe 314™ flow cell.

5 ul of Single Stranding Solution (2 ul Buffer 4 (New England BioLabs,Part No. B7004S), 8 ul Nt.BbvCI (New England BioLabs, Part No. R0632L),2 ul T7 Exonuclease (New England BioLabs, Part No. M0263L) and 8 ulnuclease-free water) was dispensed into the 314™ experimental chip andincubated for 30 minutes at room temperature, with size 4 rubber gasketscovering both ports. After incubation, 100 ul of EDS was dispensed intothe 314™ experimental chip, incubated for 1 minute at room temperature,and then removed from the 314™ flow cell. Three washes of 100 ul EDSfollowed. Next, the chip was washed three times with 100 ul AnnealingBuffer (sold as a component of the Ion PGM™ Reagents Kit, LifeTechnologies, Part No. 4468994). The annealing buffer was removed fromthe 314™ flow cell and 1 ul of Sequencing Polymerase (sold as acomponent of the Ion Sequencing Reagents Kit, Life Technologies, PartNo. 4468995) diluted 1:5 in the Annealing Buffer, was added to the 314™chip and incubated for 5 minutes at room temperature. Followingincubation, the 314™ experimental chip was placed back into the PGM™Sequencer. The second run (reverse read) was initiated and the squidwash step and wet load steps skipped.

After completion of the second run, the sequencing data for the first(forward) and second (reverse) run was obtained.

Example 5

The non-limiting, paired-end experiment described below generated tworeads for each template on sequencing beads (Ion Sphere Particles™). Oneread is called the forward read and the other, the reverse read. Sinceboth the forward and reverse reads are from the same DNA templatemolecule, the overlapping portions of the sequences are complementary.An initial pairing process determines if the forward and reverse readsoverlap. Since the read sequences are obtained from two separatereactions, the first step is to establish a potential pair. If bothsequencing reads are from the same microwell on the sequencing chip,they are considered a potential pair.

Next, the read sequences are aligned to a reference genome sequence (inthis example, E. coli genomic DNA was used). The alignment of forwardand reverse read sequences obtained from the PGM sequencing chip andcompared to the reference sequence can be performed using any availablealignment software (such as BLAST). The alignment output containsinformation about the starting and ending location of forward andreverse reads on the reference genome. This information determineswhether the forward and reverse reads overlap based on the locations onthe reference genome. The alignment output also contains informationabout mismatches, insertions, and deletions in the reads relative to thereference sequence. If the forward and reverse reads overlap,consolidation of paired-end reads occurs. This step requiresreconstructing the original single sequence from both the forward andreverse reads, only if the reads overlap. If the forward and reversereads do not overlap, reconstruction (or consolidation) of the originalsequence was not performed.

Once the forward and reverse reads are found to overlap, both reads wereconsolidated into a single sequence read. In the consolidation process,the information about each reads mismatches, insertions, and deletionsfrom alignment to the reference sequence are considered and the errorsare corrected when sufficient information is available.

A “mismatch” refers to a difference in the sequence of bases from theIon PGM™ sequencing read, as compared to the reference sequence. Forexample, if the reference sequence is an ‘A’ at a certain position, butthe corresponding position in the forward or reverse read is ‘T’, ‘C’,or ‘G’, this is called a mismatch. An “insertion error” is definedherein as a base(s) not present in the reference sequence at thecorresponding position(s); these are ‘inserted’ into the forward orreverse reads. A “deletion” is defined herein as a base ‘deleted’ fromthe forward and reverse reads, but is present in the reference sequence.The insertion and deletion errors can be collectedly called “indel”. Inthe consolidation process in the overlapped regions from both forwardand reverse reads, if the “indel” occurs only in one of the reads (butnot in the other read), the sequence bases from the non-indel read areused for the consolidated read. If both reads showed the same indel atthe same position, then the indel is retained in the consolidated read.If the indel occurs at the non-overlapped region, the indel is alsoretained in the consolidated read. The same principle applies tomismatches. For example, if a mismatch occurs only in one of the reads,but not in the other read from the pair, the sequenced base from thenon-mismatched read is retained in the consolidated read. If mismatchesoccur in both reads, or in non-overlapping regions, they are retained inthe consolidated read.

In this example, a paired-end library was prepared as disclosed inExample 4. Sequencing of forward and reverse reads was performed on aPGM™ Sequencer (Life Technologies, CA). The runs performed on the PGM™sequencer were referred to as: ULT119 and ULT120. Data from the forwardand reverse run is provided in Table 1, below.

TABLE 1 Forward reads Reverse reads Total Reads Run name ULT119 ULT120Pairable 114,868 114,868 229,736

A total of 229,736 reads were obtained. The forward and reverse runs ineach pair were adjusted to account of insertions, deletions, mismatchesand total errors against a reference DNA sample (here, E. coli).Specifically, each paired-end read was consolidated to account forvariations against the reference sequence. As a result, the number ofconsolidated reads for the above experiment was 92,665 reads. The datafrom the PGM sequencing runs can be found in FIGS. 5A, 5B, 6A, 6B, 7A,7B, 8A, 8B and 9.

FIGS. 5A and 5B disclose the total error rate for mapped reads of ULT119and ULT120 (as compared against the reference sample) as either aconsolidate read (i.e., paired end sequencing) (FIG. 5B) or as two-reads(FIG. 5A).

FIGS. 6A and 6B disclose the total deletion rate for mapped reads ofULT119 and ULT120 (as compared against the reference sample) as either aconsolidate read (i.e., paired end sequencing) (FIG. 6B) or as two-reads(FIG. 6A).

FIGS. 7A and 7B disclose the total insertion rate for mapped reads ofULT119 and ULT120 (as compared against the reference sample) as either aconsolidate read (i.e., paired end sequencing) (FIG. 7B) or as two-reads(FIG. 7A).

FIGS. 8A and 8B disclose the total mismatch rate for mapped reads ofULT119 and ULT120 (as compared against the reference sample) as either aconsolidate read (i.e., paired end sequencing) (FIG. 8B) or as two-reads(FIG. 8A).

FIG. 9 provides a graphical representation of sequencing data from runsULT119 and ULT120. As can be seen, improved sequencing accuracy isobtained when consolidating paired-end reads. Therefore, one of theadvantages of paired-end sequencing is to increase accuracy byconsolidating information from both forward and reverse sequences.

Example 6

The non-limiting, paired-end experiment described below generated tworeads for each template on sequencing beads (Ion Sphere Particles™). Thesteps for determining a pair, consolidating reads, and determining errorrates as compared to a reference sequence, were performed as outlined inExample 5.

In this example, a paired-end library was prepared as disclosed inExample 4. Sequencing of forward and reverse reads was performed on aPGM™ Sequencer. The runs performed on the PGM™ sequencer were referredto as: BUT381 and CAR321. Data from the forward and reverse runs isprovided in Table 2, below.

TABLE 2 Forward reads Reverse reads Total Reads Run name BUT381 CAR321Pairable 76,619 76,619 153,238

A total of 153,238 reads were obtained. The forward and reverse runs ineach pair were consolidated to account of insertions, deletions,mismatches and total errors against a reference DNA sample (here, E.coli). Specifically, each paired-end read was consolidated to accountfor variations against the reference sequence. As a result, the numberof consolidated reads for the above experiment was 61,295 reads. Thedata from the PGM sequencing runs can be found in FIGS. 10A, 10B, 11A,11B, 12A, 12B, 13A and 13B.

FIGS. 10A and 10B disclose the total error rate for mapped reads ofBUT381 and CAR321 (as compared against the reference sample) as either aconsolidate read (i.e., paired end sequencing) (FIG. 10B) or astwo-reads (FIG. 10A).

FIGS. 11A and 11B disclose the total deletion rate for mapped reads ofBUT381 and CAR321 (as compared against the reference sample) as either aconsolidate read (i.e., paired end sequencing) (FIG. 11B) or astwo-reads (FIG. 11A).

FIGS. 12A and 12B disclose the total insertion rate for mapped reads ofBUT381 and CAR321 (as compared against the reference sample) as either aconsolidate read (i.e., paired end sequencing) (FIG. 12B) or astwo-reads (FIG. 12A).

FIGS. 13A and 13B disclose the total mismatch rate for mapped reads ofBUT381 and CAR321 (as compared against the reference sample) as either aconsolidate read (i.e., paired end sequencing) (FIG. 13B) or astwo-reads (FIG. 13A).

Table 3 provides a summary of both experiments disclosed in Examples 5and 6. As can be seen, an improved accuracy is obtained whenconsolidating paired-end reads. Therefore, one advantage of paired-endsequencing is to increase sequencing accuracy by consolidatinginformation from both forward and reverse sequences. Overall, a 3-4 foldincrease in sequencing accuracy was observed in these experiments whenconsolidating paired-end reads.

TABLE 3 As 2 reads Consolidated As 2 reads Consolidated Total Base20,372,929 10,300,696 13,448,227 7,240,160 Deletion 163,472 15,98882,084 14,178 Insertion 175,237 8,782 76,168 4,397 Mismatch 195,98450,544 105,428 32,176 Total Error 534,693 75,314 263,680 50,751 BaseAccuracy 97.4% 99.3% 98% 99.3% Overall, 3-4 fold increase in accuracy

Example 7 Variant Assessment

In this example, the ability for variant calling after error correctionand consolidation was assessed using an exemplary paired end sequencingmethod according to the disclosure. A total of 6,263 substitutions weresimulated into an E. coli reference sample (here, a DH10B referencesample). Sequencing runs were performed on a PGM™ sequencer using an IonChip, according to Example 4. Once the forward and reverse runs werecomplete, the runs were consolidated to obtain paired-end informationusing the simulated substitution E. coli sample as the reference sample.The consolidation process of both the forward and reverse runs wasperformed as described in Examples 5 and 6.

Table 4 provides a summary of the PGM™ run data obtained for UTL119 andUTL120 using the simulated substituted reference sample. Table 5provides a summary of the PGM™ run data obtained for BUT381 and CAR321using the simulated substituted reference sample.

As can be seen, the consolidation process in both independentexperiments retained the ability for variant calling.

TABLE 4 ULT119 & ULT120 Number of Bases Percent Avg Coverage No Coverage1174 0 Non Ref 3158 62% 3.08 As Ref 1931 38% 1.44

TABLE 5 BUT381 & CAR321 Number of Bases Percent Avg Coverage No Coverage1990 0 Non Ref 2283 53% 2.70 As Ref 1990 47% 1.36

Example 8

In this exemplary example, a paired-end library was prepared as follows:

Prepare the Paired-End Adaptor Mixture

Paired-End Adaptor oligonucleotides were created to form a paired-endadapter mixture for use in the library preparation. Paired-end P1Adaptor oligonucleotide 1 and 2 contain an Nt.BbvCl nick site, whileAdaptor A oligonucleotides 1 and 2 complete the adaptors mixture. Alloligonucleotides were HPLC purified and subjected to sodium saltexchange.

Paired-end P1 5′-CCTCTCTATGGGCAGTCG Adapter oligo 1 GTGATCCTCAGC-3′SEQ ID NO: 1 Paired-end P1 5′-GCTGAGGATCACCGACTG Adapter oligo 2CCCATAGAGAGGTT-3′ SEQ ID NO: 3 Adapter A 5′-CCATCTCATCCCTGCGTG oligo 1TCTCCGACTCAG-3′ SEQ ID NO: 4 Adapter A 5′-CTGAGTCGGAGACACGCA oligo 2GGGATGAGATGG*T*T-3′ SEQ ID NO: 5

The following paired-end sequencing primer was ordered and purified asabove.

5′-C*C*A*T*CTCATCCCTGCGTGTCTCCGAC-3′, wherein * denotes aphosphorothioate bond. (SEQ ID NO: 6)

Prepare Enzyme Denaturation Solution

An enzyme denaturing solution (EDS) was prepared as follows for use inthe paired-end library. 1.3 ml of EDS was used per sequencing reaction:TE pH 8.0; 2% SDS; 50 mM NaCl.

Prepare the Paired-End Adaptors Mixture

Each Adaptor oligonucleotide was diluted to a concentration of 100 umwith Y μl of nuclease-free water, determined as follows:X nmole oligo/100 nmole×1000=Y μl of nuclease-free water

The following reactions were prepared in separate sterile tubes:

Tube 1 Tube 2 Paired-end P1 Adapter oligo 1 (100 μM) 50 μL — Paired-endP1 Adapter oligo 2 (100 μM) 50 μL — A Adapter oligo 1 (100 μM) — 50 μL AAdapter oligo 2 (100 μM) — 50 μL T4 DNA Ligase Buffer (5X) 25 μL 25 μL

Each tube was heated using a thermal cycler as follows:

A) Heat at 90° C. for 2 minutes

B) Cool to room temperature (˜120 minutes)

Equal volumes of each adaptor were combined into a single tube to form acomplete adaptor mixture at a final concentration of about 20 μM. Thecomplete adaptor mixture was then stored at −20° C. until ready toprogress to the next step.

Library Preparation: Substitute the Paired-End Adaptors Mixture

A non-barcoded library was prepared as essentially described in the IonXpress™ Plus gDNA and Amplicon Library Preparation User Guide (LifeTechnologies, Part No. 4471989), except the standard adaptors of the IonXpress™ Plus Library Kit were substituted with the adaptor mixtureprepared above. The library was prepared essentially according to theprotocol outlined in the above Library Preparation User Guide, which isincorporated herein in its entirety.

Perform Standard Forward Sequencing

A standard sequencing protocol was performed as essentially described inthe Ion Sequencing 200 Kit User Guide (Life Technologies, Part No.4471998), which is incorporated herein in its entirety, except that thestandard sequencing primer was substituted for the paired-end sequencingprimer, prepared above. The paired-end sequencing primer was added tothe Ion Sphere Particles (ISPs) as follows:

Volume per chip Component Ion 314 ™ Chip Ion 316 ™ Chip Paired-endSequencing Primer 5 μL 12 μL Enriched ISPs in Annealing Buffer 8 μL 15μL Total volume 13 μL  27 μL

The standard sequencing protocol was then performed as essentiallyoutlined in the Sequencing Kit User Guide.

Denature the Sequencing Polymerase

After the sequencing run was complete, the Ion chip was removed from thePGM™ System and placed on a grounding plate or in an Ion centrifugeadapter/rotor bucket. While the Ion chip was removed, a dummy (used)chip was clamped into the PGM.

100 μL of EDS was applied into the loading port on the Ion chip. TheChip was then incubated at room temperature for 1 minute. Afterincubation, the chip was washed three times with 100 μL of EDS: for eachwash, 100 μL of EDS was added to the loading port and then removed.

A 1× NEBuffer 2 was prepared by diluting the stock buffer 1:10 withnuclease-free water. The chip was then washed three times with 100 μL of1× NEBuffer 2.

Fill in the Sequence

An extension solution was prepared as follows:

Volume per chip Component Ion 314 ™ Chip Ion 316 ™ Chip Nuclease-freewater 14 μL  56 μL  10 mM dNTPs (prepare a 1:4 2 μL 8 μL dilution ofstock dNTPs from the Ion Xpress ™ Template 200 Kit) 10X NEBuffer 2 2 μL8 μL DNA Polymerase I, Large 2 μL 2 μL (Klenow) Fragment Total 20 μL  80μL 

The following volumes of extension solution were applied to the loadingport of the respective chip.

Ion 314™ Chip: 15 μl (˜5 μl overflow in flow cell wells)

Ion 316™ Chip: 75 μl (˜25 μl overflow in flow cell wells)

The chip was then transferred to a covered heating block (at 25° C.)containing a 50 ml tube cap filled with deionized water (to preventevaporation) and incubated for 10 minutes. After which, 100 ul of EDSwas loaded into the loading port, and incubated at room temperature for1 minute. After incubation, as much liquid as possible was removed fromthe loading port. The chip was then washed 3 times with 100 μl of EDS. A1× NEBuffer 4 was prepared by diluting the stock buffer 1:10 withnuclease-free water. The chip was then washed three times with 100 μL of1× NEBuffer 4.

Denature the Template

A second strand solution was prepared as outlined below.

Volume per chip Component Ion 314 ™ Chip Ion 316 ™ Chip Nuclease-freewater 42 μL  168 μL  NEBuffer 4 (1x) 6 μL 24 μL Nt.BbvCl 6 μL 24 μL T7Exonuclease 6 μL 24 μL Total 60 μL  240 μL 

The following amounts of the second strand solution was dispensed intothe respective Chip.

Ion 314™ Chip: 15 μl (˜5 μl overflow in flow cell wells)

Ion 316™ Chip: 75 μl (˜25 μl overflow in flow cell wells)

The chip was placed on a 1.5 ml freezer rack half filled with deionizedwater and incubated at room temperature for 20 minutes. The fluid in thechip was replaced from the loading port, and the second strand solutionwas re-applied to the chip for a second and third time, with therespective incubation times for a total incubation time of 60 minutes.After the final incubation, residual liquid was removed from the chipand 100 μl of EDs was applied to the loading port. The chip wasincubated at room temperature for 1 minute, after which the EDS solutionwas removed from the chip. The chip was subsequently washed 3 times with100 μl of EDS.

The chip was then washed 3 times with 100 μl of annealing buffer fromthe Ion Sequencing 200 Kit.

The sequencing polymerase from the Ion Sequencing 200 Kit was diluted inannealing buffer as shown below:

Volume per chip Component Ion 314 ™ Chip Ion 316 ™ Chip SequencingPolymerase 1.5 μL  6 μL Annealing Buffer  6 μL 24 μL Total volume 7.5 μL30 μL

Any residual annealing buffer from the wash steps was removed from thechip, and the diluted sequencing polymerase was applied (in full volume)to the loading port and incubated at room temperature for 5 minutes.During incubation, the sequencing key for the PGM system was altered asshown below:

A) From the main screen, press Options.

B) Press Advanced.

C) Press Change “Library Key Sequence”.

D) Enter the new key sequence: TCAGC.

E) Press Back to return to the main screen.

Following incubation, the dummy (used) chip was removed from the PGMsystem and the prepared paired-end sequencing chip was loaded. Thesequencing experiment was initiated via pressing “experiment” on the PGMsystem. During the chip check, the “wetload” box was unchecked. The chipwashing and loading steps provided in the Ion Sequencing 200 Kit werealso obviated to proceed directly to selecting the experimentalconfiguration and performance of the sequencing run.

Example 9

In this exemplary embodiment, a paired-end library was prepared asfollows:

Prepare the Paired-End Adaptor Mixture

Paired-End Adaptor oligonucleotides were created to form a paired-endadapter mixture for use in the library preparation. Paired-end P1Adaptor oligonucleotide 3 and 4 contain an Nt.BbvCl nick site, whileAdaptor A oligonucleotides 3 and 4 complete the adaptors mixture. Alloligonucleotides were HPLC purified and subjected to sodium saltexchange. In the oligonucleotides * denotes a phosphorothioate bond. Inthe oligonucleotides “Y” denotes a C or a T nucleotide at that position.

Paired-end P1 5′-CCACTACGCCTCCGCTTT Adapter oligo 3CCTCTCTATGGGCAGTCGGTG ATCCTCAGC-3′ SEQ ID NO: 7 Paired-end P15′-GCTGAGGATCACCGACTG Adapter oligo 4 CCCATAGAGAGGAAAGCGGAGGCGTAGYGG*T*T-3′ SEQ ID NO: 8 Adapter A 5′-CCATCTCATCCCTGCGTG oligo 3TCTCCGACTCAG-3′ SEQ ID NO: 4 Adapter A 5′-CTGAGTCGGAGACACGCA oligo 4GGGATGAGATGG*T*T-3′ SEQ ID NO: 5

The following paired-end sequencing primer was ordered and purified asabove.

5′-C*C*A*T*CTCATCCCTGCGTGTCTCCGAC-3′ (SEQ ID NO: 6), wherein * denotes aphosphorothioate bond.

Prepare Enzyme Denaturation Solution

An enzyme denaturing solution (EDS) was prepared as follows for use inthe paired-end library: 10×TE pH 8.0 (5 ml), 20% SDS (5 ml) and 50 mMNaCl (0.5 ml) to a total of 50 ml in nuclease-free water. Finalconcentration: 1×TE pH 8.0, 2% SDS and 50 mM NaCl. 1.3 ml of EDS wasused per sequencing reaction.

Prepare the Paired-End Adaptors Mixture

Each Adaptor oligonucleotide was diluted to a concentration of 100 umwith Y μl of nuclease-free water, determined as follows:X nmole oligo/100 nmole×1000=Y μl of nuclease-free water

The following reactions were prepared in separate sterile tubes:

Tube 1 Tube 2 Paired-end P1 Adapter oligo 3 (100 μM) 50 μL — Paired-endP1 Adapter oligo 4 (100 μM) 50 μL — A Adapter oligo 3 (100 μM) — 50 μL AAdapter oligo 4 (100 μM) — 50 μL T4 DNA Ligase Buffer (5X) 25 μL 25 μL

Each tube was heated using a thermal cycler as follows:

A) Heat at 90° C. for 2 minutes

B) Cool to room temperature (˜120 minutes)

Equal volumes of each adaptor were combined into a single tube to form acomplete adaptor mixture at a final concentration of about 20 μM. Thecomplete adaptor mixture was then stored at −20° C. until ready toprogress to the next step.

Library Preparation: Substitute the Paired-End Adaptors Mixture

A non-barcoded library was prepared as essentially described in the IonPlus Fragment Library Kit (Life Technologies, Part No. 4471252), exceptthe standard adaptors of the Ion Plus Fragment Library Kit weresubstituted with the adaptor mixture prepared above. The library wasprepared essentially according to the protocol outlined in the aboveLibrary Preparation User Guide, which is incorporated herein in itsentirety.

Template Preparation

A standard template protocol was performed as essentially described inthe Ion Template Kit User Guide (Life Technologies, Part No. 4469004),which is incorporated herein in its entirety.

Perform Standard Forward Sequencing

A standard sequencing protocol was performed as essentially described inthe Ion Sequencing 200 Kit User Guide (Life Technologies, Part No.4471998), which is incorporated herein in its entirety, except that thestandard sequencing primer was substituted for the paired-end sequencingprimer, prepared above. The paired-end sequencing primer was added tothe Ion Sphere Particles (ISPs) as follows:

Volume per chip Component Ion 314 ™ Chip Ion 316 ™ Chip Paired-endSequencing Primer 5 μL 12 μL Enriched ISPs in Annealing Buffer 8 μL 15μL Total volume 13 μL  27 μL

The standard sequencing protocol was then performed as essentiallyoutlined in the Sequencing Kit User Guide.

Denature the Sequencing Polymerase

After the sequencing run was complete, the Ion chip was removed from thePGM™ System and placed on a grounding plate or in an Ion centrifugeadapter/rotor bucket. While the Ion chip was removed, a dummy (used)chip was clamped into the PGM.

100 μL of EDS was applied into the loading port on the Ion chip. TheChip was then incubated at room temperature for 1 minute. Afterincubation, the chip was washed once with 100 μL of EDS; the EDS wasadded to the loading port and then removed.

A 1× NEBuffer 2 was prepared by diluting the stock buffer 1:10 withnuclease-free water. The chip was then washed three times with 100 μL of1× NEBuffer 2.

Fill in the Sequence

An extension solution was prepared as follows:

Volume per chip Component Ion 314 ™ Chip Ion 316 ™ Chip Nuclease-freewater 14 μL  56 μL  10 mM dNTPs (prepare a 1:4 2 μL 8 μL dilution ofstock dNTPs from the Ion Xpress ™ Template 200 Kit) 10X NEBuffer 2 2 μL8 μL DNA Polymerase I, Large 2 μL 2 μL (Klenow) Fragment Total 20 μL  80μL 

The following volumes of extension solution were applied to the loadingport of the respective chip.

Ion 314™ Chip: 15 μl (˜5 μl overflow in flow cell wells)

Ion 316™ Chip: 75 μl (˜25 μl overflow in flow cell wells)

The chip was then transferred to a covered heating block (at 25° C.)containing a 50 ml tube cap filled with deionized water (to preventevaporation) and incubated for 10 minutes. After which, 100 ul of EDSwas loaded into the loading port, and incubated at room temperature for1 minute. After incubation, as much liquid as possible was removed fromthe loading port. A 1× NEBuffer 4 was prepared by diluting the stockbuffer 1:10 with nuclease-free water. The chip was then washed threetimes with 100 μL of 1× NEBuffer 4.

Denature the Template

A second strand solution was prepared as outlined below.

Volume per chip Component Ion 314 ™ Chip Ion 316 ™ Chip Nuclease-freewater 42 μL  168 μL  NEBuffer 4 (1x) 6 μL 24 μL Nt.BbvCl 6 μL 24 μL T7Exonuclease 6 μL 24 μL Total 60 μL  240 μL 

The following amounts of the second strand solution was dispensed intothe respective Chip.

Ion 314™ Chip: 15 μl (˜5 μl overflow in flow cell wells)

Ion 316™ Chip: 75 μl (˜25 μl overflow in flow cell wells)

The chip was placed on a 1.5 ml freezer rack half filled with deionizedwater and incubated at room temperature for 20 minutes. The fluid in thechip was replaced from the loading port, and the second strand solutionwas re-applied to the chip for a second and third time, with therespective incubation times for a total incubation time of 60 minutes.After the final incubation, residual liquid was removed from the chipand 100 μl of EDS was applied to the loading port. The chip was thenwashed 3 times with 100 μl of annealing buffer from the Ion Sequencing200 Kit.

The sequencing polymerase from the Ion Sequencing 200 Kit was diluted inannealing buffer as shown below:

Volume per chip Component Ion 314 ™ Chip Ion 316 ™ Chip SequencingPolymerase 1.5 μL  6 μL Annealing Buffer  6 μL 24 μL Total volume 7.5 μL30 μL

Any residual annealing buffer from the wash steps was removed from thechip, and the diluted sequencing polymerase was applied (in full volume)to the loading port and incubated at room temperature for 5 minutes.During incubation, the sequencing key for the PGM system was altered asshown below:

F) From the main screen, press Options.

G) Press Advanced.

H) Press Change “Library Key Sequence”.

I) Enter the new key sequence: TCAGC.

J) Press Back to return to the main screen.

Following incubation, the dummy (used) chip was removed from the PGMsystem and the prepared paired-end sequencing chip was loaded. Thesequencing experiment was initiated via pressing “experiment” on the PGMsystem. During the chip check, the “wetload” box was unchecked. The chipwashing and loading steps provided in the Ion Sequencing 200 Kit werealso obviated to proceed directly to selecting the experimentalconfiguration and performance of the sequencing run.

Example 10

In this non-limiting example, a paired-end library was prepared asfollows:

Prepare the Paired-End Adaptor Mixture

Paired-End Adaptor oligonucleotides were created to form a paired-endadapter mixture for use in the library preparation. Paired-end P1Adaptor oligonucleotide 3 and 4 contain an Nt.BbvCl nick site, whileAdaptor A oligonucleotides 3 and 4 complete the adaptors mixture. Alloligonucleotides were HPLC purified and subjected to sodium saltexchange. In the oligonucleotides “Y” denotes a C or a T nucleotide atthat position.

Paired-end P1 5′-CCACTACGCCTCCGCTTT Adapter oligo 3CCTCTCTATGGGCAGTCGGTG ATCCTCAGC-3′ SEQ ID NO: 7 Paired-end P15′-GCTGAGGATCACCGACTG Adapter oligo 4 CCCATAGAGAGGAAAGCGGAGGCGTAGYGG*T*T-3′ SEQ ID NO: 8 Adapter A 5′-CCATCTCATCCCTGCGTG oligo 3TCTCCGACTCAG-3′ SEQ ID NO: 4 Adapter A 5′-CTGAGTCGGAGACACGCA oligo 4GGGATGAGATGG*T*T-3′ SEQ ID NO: 5

The following paired-end sequencing primer was ordered and purified asabove.

5′-C*C*A*T*CTCATCCCTGCGTGTCTCCGAC-3′ (SEQ ID NO: 6), wherein * denotes aphosphorothioate bond. In the oligonucleotides * denotes aphosphorothioate bond.

Prepare Enzyme Denaturation Solution

An enzyme denaturing solution (EDS) was prepared as follows for use inthe paired-end library: 10×TE pH 8.0 (5 ml), 20% SDS (5 ml) and 50 mMNaCl (0.5 ml) to a total of 50 ml in nuclease-free water. Finalconcentration: 1×TE pH 8.0, 2% SDS and 50 mM NaCl. 1.3 ml of EDS wasused per sequencing reaction.

Prepare the Paired-End Adaptors Mixture

Each Adaptor oligonucleotide was diluted to a concentration of 100 umwith Y μl of nuclease-free water, determined as follows:X nmole oligo/100 nmole×1000=Y μl of nuclease-free water

The following reactions were prepared in separate sterile tubes:

Tube 1 Tube 2 Paired-end P1 Adapter oligo 3 (100 μM) 50 μL — Paired-endP1 Adapter oligo 4 (100 μM) 50 μL — A Adapter oligo 3 (100 μM) — 50 μL AAdapter oligo 4 (100 μM) — 50 μL T4 DNA Ligase Buffer (5X) 25 μL 25 μL

Each tube was heated using a thermal cycler as follows:

A) Heat at 90° C. for 2 minutes

B) Cool to room temperature (˜120 minutes)

Equal volumes of each adaptor were combined into a single tube to form acomplete adaptor mixture at a final concentration of about 20 μM. Thecomplete adaptor mixture was then stored at −20° C. until ready toprogress to the next step.

Library Preparation: Substitute the Paired-End Adaptors Mixture

A non-barcoded library was prepared as essentially described in the IonPlus Fragment Library Kit (Life Technologies, Part No. 4471252), exceptthe standard adaptors of the Ion Plus Fragment Library Kit weresubstituted with the adaptor mixture prepared above. The library wasprepared essentially according to the protocol outlined in the aboveLibrary Preparation User Guide, which is incorporated herein in itsentirety.

Template Preparation

A standard template protocol was performed as essentially described inthe Ion Template Kit User Guide (Life Technologies, Part No. 4469004),which is incorporated herein in its entirety.

Perform Standard Forward Sequencing

A standard sequencing protocol was performed as essentially described inthe Ion Sequencing 200 Kit User Guide (Life Technologies, Part No.4471998), which is incorporated herein in its entirety, except that thestandard sequencing primer was substituted for the paired-end sequencingprimer, prepared above. The paired-end sequencing primer was added tothe Ion Sphere Particles (ISPs) as follows:

Volume per chip Component Ion 314 ™ Chip Ion 316 ™ Chip Paired-endSequencing Primer 5 μL 12 μL Enriched ISPs in Annealing Buffer 8 μL 15μL Total volume 13 μL  27 μL

The standard sequencing protocol was then performed as essentiallyoutlined in the Sequencing Kit User Guide.

Denature the Sequencing Polymerase

After the sequencing run was complete, the Ion chip was removed from thePGM™ System and placed on a grounding plate or in an Ion centrifugeadapter/rotor bucket. While the Ion chip was removed, a dummy (used)chip was clamped into the PGM.

100 μL of EDS was applied into the loading port on the Ion chip. TheChip was then incubated at room temperature for 1 minute.

A 1× NEBuffer 2 was prepared by diluting the stock buffer 1:10 withnuclease-free water. The chip was then washed three times with 100 μL of1× NEBuffer 2.

Fill in the Sequence

An extension solution was prepared as follows:

Volume per chip Ion 314 ™ Ion 316 ™ Component Chip Chip Nuclease-freewater 14 μL  56 μL  10 mM dNTPs (prepare a 1:4 dilution 2 μL 8 μL ofstock dNTPs from the Ion Xpress ™ Template 200 Kit) 10X NEBuffer 2 2 μL8 μL DNA Polymerase I, Large (Klenow) 2 μL 2 μL Fragment Total 20 μL  80μL 

The following volumes of extension solution were applied to the loadingport of the respective chip.

Ion 314™ Chip: 15 μl (˜5 μl overflow in flow cell wells)

Ion 316™ Chip: 75 μl (˜25 μl overflow in flow cell wells)

The chip was then transferred to a covered heating block (at 25° C.)containing a 50 ml tube cap filled with deionized water (to preventevaporation) and incubated for 10 minutes. After which, 100 ul of EDSwas loaded into the loading port, and incubated at room temperature for1 minute. After incubation, as much liquid as possible was removed fromthe loading port. A 1× NEBuffer 4 was prepared by diluting the stockbuffer 1:10 with nuclease-free water. The chip was then washed threetimes with 100 μL of 1× NEBuffer 4.

Denature the Template

A second strand solution was prepared as outlined below.

Volume per chip Ion 314 ™ Ion 316 ™ Component Chip Chip Nuclease-freewater 42 μL  168 μL  NEBuffer 4 (1x) 6 μL 24 μL Nt.BbvCl 6 μL 24 μL T7Exonuclease 6 μL 24 μL Total 60 μL  240 μL 

The following amounts of the second strand solution was dispensed intothe respective Chip.

Ion 314™ Chip: 15 μl (˜5 μl overflow in flow cell wells)

Ion 316™ Chip: 75 μl (˜25 μl overflow in flow cell wells)

The chip was placed on a 1.5 ml freezer rack half filled with deionizedwater and incubated at room temperature for 20 minutes. The fluid in thechip was replaced from the loading port, and the second strand solutionwas re-applied to the chip for a second and third time, with therespective incubation times for a total incubation time of 60 minutes.After the final incubation, residual liquid was removed from the chipand 100 μl of EDS was applied to the loading port. The chip wasincubated at room temperature for 1 minute, after which the EDS solutionwas removed from the chip.

The chip was then washed 3 times with 100 μl of annealing buffer fromthe Ion Sequencing 200 Kit.

The sequencing polymerase from the Ion Sequencing 200 Kit was diluted inannealing buffer as shown below:

Volume per chip Component Ion 314 ™ Chip Ion 316 ™ Chip SequencingPolymerase 1.5 μL  6 μL Annealing Buffer   6 μL 24 μL Total volume 7.5μL 30 μL

Any residual annealing buffer from the wash steps was removed from thechip, and the diluted sequencing polymerase was applied (in full volume)to the loading port and incubated at room temperature for 5 minutes.During incubation, the sequencing key for the PGM system was altered asshown below:

K) From the main screen, press Options.

L) Press Advanced.

M) Press Change “Library Key Sequence”.

N) Enter the new key sequence: TCAGC.

O) Press Back to return to the main screen.

Following incubation, the dummy (used) chip was removed from the PGMsystem and the prepared paired-end sequencing chip was loaded. Thesequencing experiment was initiated via pressing “experiment” on the PGMsystem. During the chip check, the “wetload” box was unchecked. The chipwashing and loading steps provided in the Ion Sequencing 200 Kit werealso obviated to proceed directly to selecting the experimentalconfiguration and performance of the sequencing run.

Example 11

In this non-limiting example, a paired-end library was prepared asfollows.

Prepare the Paired-End Adaptor Mixture

Paired-End Adaptor oligonucleotides were created to form a paired-endadapter mixture for use in the library preparation. Paired-end P1Adaptor oligonucleotide 3 and 4 contain an Nt.BbvCl nick site, whileAdaptor A oligonucleotides 3 and 4 complete the adaptors mixture. Alloligonucleotides were HPLC purified and subjected to sodium saltexchange. In the oligonucleotides * denotes a phosphorothioate bond. Inthe oligonucleotides “Y” denotes a C or a T nucleotide at that position.

Paired-end P1 5′-CCACTACGCCTCCGCTTT Adapter oligo 3CCTCTCTATGGGCAGTCGGTG ATCCTCAGC-3′ SEQ ID NO: 7 Paired-end P15′-GCTGAGGATCACCGACTG Adapter oligo 4 CCCATAGAGAGGAAAGCGGAGGCGTAGYGG*T*T-3′ SEQ ID NO: 8 Adapter A 5′-CCATCTCATCCCTGCGTG oligo 3TCTCCGACTCAG-3′ SEQ ID NO: 4 Adapter A 5′-CTGAGTCGGAGACACGCA oligo 4GGGATGAGATGG*T*T-3′ SEQ ID NO: 5

The following paired-end sequencing primer was ordered and purified asabove.

5′-C*C*A*T*CTCATCCCTGCGTGTCTCCGAC-3′ (SEQ ID NO: 6), wherein * denotes aphosphorothioate bond.

Prepare Enzyme Denaturation Solution

An enzyme denaturing solution (EDS) was prepared as follows for use inthe paired-end library: 10×TE pH 8.0 (5 ml), 20% SDS (5 ml) and 50 mMNaCl (0.5 ml) to a total of 50 ml in nuclease-free water. Finalconcentration: 1×TE pH 8.0, 2% SDS and 50 mM NaCl. 1.3 ml of EDS wasused per sequencing reaction.

Prepare Additive Solution

A 4% or 8% polyvinylpyrrolidone solution (PVP) was prepared as followsfor use in the paired-end library. Dissolve 0.4 grams of PVP40 into 4.8ml of nuclease-free water to a total volume of 5 ml (8% solution) ordissolve 0.2 grams of PVP40 into 4.8 ml of nuclease-free water to atotal volume of 5 ml (4% solution).

Prepare the Paired-End Adaptors Mixture

Each Adaptor oligonucleotide was diluted to a concentration of 100 umwith Y μl of nuclease-free water, determined as follows:X nmole oligo/100 nmole×1000=Y μl of nuclease-free water

The following reactions were prepared in separate sterile tubes:

Tube 1 Tube 2 Paired-end P1 Adapter oligo 3 (100 μM) 50 μL — Paired-endP1 Adapter oligo 4 (100 μM) 50 μL — A Adapter oligo 3 (100 μM) — 50 μL AAdapter oligo 4 (100 μM) — 50 μL T4 DNA Ligase Buffer (5X) 25 μL 25 μL

Each tube was heated using a thermal cycler as follows:

A) Heat at 90° C. for 2 minutes

B) Cool to room temperature (˜120 minutes)

Equal volumes of each adaptor were combined into a single tube to form acomplete adaptor mixture at a final concentration of about 20 μM. Thecomplete adaptor mixture was then stored at −20° C. until ready toprogress to the next step.

Library Preparation: Substitute the Paired-End Adaptors Mixture

A non-barcoded library was prepared as essentially described in the IonPlus Fragment Library Kit (Life Technologies, Part No. 4471252), exceptthe standard adaptors of the Ion Plus Fragment Library Kit weresubstituted with the adaptor mixture prepared above. The library wasprepared essentially according to the protocol outlined in the aboveLibrary Preparation User Guide, which is incorporated herein in itsentirety.

Template Preparation

A standard template protocol was performed as essentially described inthe Ion Template Kit User Guide (Life Technologies, Part No. 4469004),which is incorporated herein in its entirety.

Perform Standard Forward Sequencing

A standard sequencing protocol was performed as essentially described inthe Ion Sequencing 200 Kit User Guide (Life Technologies, Part No.4471998), which is incorporated herein in its entirety, except that thestandard sequencing primer was substituted for the paired-end sequencingprimer, prepared above. The paired-end sequencing primer was added tothe Ion Sphere Particles (ISPs) as follows:

Volume per chip Component Ion 314 ™ Chip Ion 316 ™ Chip Paired-endSequencing Primer 5 μL 12 μL Enriched ISPs in Annealing Buffer 8 μL 15μL Total volume 13 μL  27 μL

The standard sequencing protocol was then performed as essentiallyoutlined in the Sequencing Kit User Guide.

Denature the Sequencing Polymerase

After the sequencing run was complete, the Ion chip was removed from thePGM™ System and placed on a grounding plate or in an Ion centrifugeadapter/rotor bucket. While the Ion chip was removed, a dummy (used)chip was clamped into the PGM.

100 μL of EDS was applied into the loading port on the Ion chip. TheChip was then incubated at room temperature for 1 minute.

A 1× NEBuffer 2 was prepared by diluting the stock buffer 1:10 withnuclease-free water. An additive, PVP, was also added to the buffer to afinal concentration of 0.4%. The chip was then washed three times with100 μL of 1× NEBuffer 2 containing PVP.

Fill in the Sequence

An extension solution was prepared as follows:

Volume per chip Ion 314 ™ Ion 316 ™ Component Chip Chip Nuclease-freewater 12 μL  48 μL  10 mM dNTPs (prepare a 1:4 dilution 2 μL 8 μL ofstock dNTPs from the Ion Xpress ™ Template 200 Kit) PVP 0.4% 2 μL 8 μL10X NEBuffer 2 2 μL 8 μL DNA Polymerase I, Large (Klenow) 2 μL 8 μLFragment Total 20 μL  80 μL 

The following volumes of extension solution were applied to the loadingport of the respective chip.

Ion 314™ Chip: 15 μl (˜5 μl overflow in flow cell wells)

Ion 316™ Chip: 75 μl (˜25 μl overflow in flow cell wells)

The chip was then transferred to a covered heating block (at 25° C.)containing a 50 ml tube cap filled with deionized water (to preventevaporation) and incubated for 10 minutes. After which, 100 ul of EDSwas loaded into the loading port, and incubated at room temperature for1 minute. After incubation, as much liquid as possible was removed fromthe loading port. A 1× NEBuffer 4 was prepared by diluting the stockbuffer 1:10 with nuclease-free water. An additive, PVP, was also addedto the buffer to a final concentration of 0.4%. The chip was then washedthree times with 100 μL of 1× NEBuffer 4 containing PVP.

Denature the Template

A second strand solution was prepared as outlined below.

Volume per chip Ion 314 ™ Ion 316 ™ Component Chip Chip Nuclease-freewater 42 μL  168 μL  NEBuffer 4 (1x) 6 μL 24 μL Nt.BbvCl 6 μL 24 μL T7Exonuclease 6 μL 24 μL Total 60 μL  240 μL 

The following amounts of the second strand solution was dispensed intothe respective Chip.

Ion 314™ Chip: 15 μl (˜5 μl overflow in flow cell wells)

Ion 316™ Chip: 75 μl (˜25 μl overflow in flow cell wells)

The chip was placed on a 1.5 ml freezer rack half filled with deionizedwater and incubated at room temperature for 20 minutes. The fluid in thechip was replaced from the loading port, and the second strand solutionwas re-applied to the chip for a second and third time, with therespective incubation times for a total incubation time of 60 minutes.After the final incubation, residual liquid was removed from the chipand 100 μl of EDS was applied to the loading port. The chip wasincubated at room temperature for 1 minute, after which the EDS solutionwas removed from the chip.

The chip was then washed 3 times with 100 μl of annealing buffer fromthe Ion Sequencing 200 Kit.

The sequencing polymerase from the Ion Sequencing 200 Kit was diluted inannealing buffer as shown below:

Volume per chip Component Ion 314 ™ Chip Ion 316 ™ Chip SequencingPolymerase 1.5 μL  6 μL Annealing Buffer   6 μL 24 μL Total volume 7.5μL 30 μL

Any residual annealing buffer from the wash steps was removed from thechip, and the diluted sequencing polymerase was applied (in full volume)to the loading port and incubated at room temperature for 5 minutes.During incubation, the sequencing key for the PGM system was altered asshown below:

P) From the main screen, press Options.

Q) Press Advanced.

R) Press Change “Library Key Sequence”.

S) Enter the new key sequence: TCAGC.

T) Press Back to return to the main screen.

Following incubation, the dummy (used) chip was removed from the PGMsystem and the prepared paired-end sequencing chip was loaded. Thesequencing experiment was initiated via pressing “experiment” on the PGMsystem. During the chip check, the “wetload” box was unchecked. The chipwashing and loading steps provided in the Ion Sequencing 200 Kit werealso obviated to proceed directly to selecting the experimentalconfiguration and performance of the sequencing run.

Example 12

In this non-limiting example, a paired-end library was prepared asfollows.

Prepare the Paired-End Adaptor Mixture

Paired-End Adaptor oligonucleotides were created to form a paired-endadapter mixture for use in the library preparation. Paired-end P1Adaptor oligonucleotide 3 and 4 contain an Nt.BbvCl nick site, whileAdaptor A oligonucleotides 3 and 4 complete the adaptors mixture. Alloligonucleotides were HPLC purified and subjected to sodium saltexchange. In the oligonucleotides * denotes a phosphorothioate bond. Inthe oligonucleotides “Y” denotes a C or a T nucleotide at that position.

Paired-end P1 5′-CCACTACGCCTCCGCTTT Adapter oligo 3CCTCTCTATGGGCAGTCGGTG ATCCTCAGC-3′ SEQ ID NO: 7 Paired-end P15′-GCTGAGGATCACCGACTG Adapter oligo 4 CCCATAGAGAGGAAAGCGGAGGCGTAGYGG*T*T-3′ SEQ ID NO: 8 Adapter A 5′-CCATCTCATCCCTGCGTG oligo 3TCTCCGACTCAG-3′ SEQ ID NO: 4 Adapter A 5′-CTGAGTCGGAGACACGCA oligo 4GGGATGAGATGG*T*T-3′ SEQ ID NO: 5

The following paired-end sequencing primer was ordered and purified asabove.

5′-C*C*A*T*CTCATCCCTGCGTGTCTCCGAC-3′ (SEQ ID NO: 6), wherein * denotes aphosphorothioate bond.

Prepare Enzyme Denaturation Solution

An enzyme denaturing solution (EDS) was prepared as follows for use inthe paired-end library: 10×TE pH 8.0 (5 ml), 20% SDS (5 ml) and 50 mMNaCl (0.5 ml) to a total of 50 ml in nuclease-free water. Finalconcentration: 1×TE pH 8.0, 2% SDS and 50 mM NaCl. 1.3 ml of EDS wasused per sequencing reaction.

Prepare the Paired-End Adaptors Mixture

Each Adaptor oligonucleotide was diluted to a concentration of 100 umwith Y μl of nuclease-free water, determined as follows:X nmole oligo/100 nmole×1000=Y μl of nuclease-free water

The following reactions were prepared in separate sterile tubes:

Tube 1 Tube 2 Paired-end P1 Adapter oligo 3 (100 μM) 50 μL — Paired-endP1 Adapter oligo 4 (100 μM) 50 μL — A Adapter oligo 3 (100 μM) — 50 μL AAdapter oligo 4 (100 μM) — 50 μL T4 DNA Ligase Buffer (5X) 25 μL 25 μL

Each tube was heated using a thermal cycler as follows:

A) Heat at 90° C. for 2 minutes

B) Cool to room temperature (˜120 minutes)

Equal volumes of each adaptor were combined into a single tube to form acomplete adaptor mixture at a final concentration of about 20 μM. Thecomplete adaptor mixture was then stored at −20° C. until ready toprogress to the next step.

Library Preparation: Substitute the Paired-End Adaptors Mixture

A non-barcoded library was prepared as essentially described in the IonPlus Fragment Library Kit (Life Technologies, Part No. 4471252), exceptthe standard adaptors of the Ion Plus Fragment Library Kit weresubstituted with the adaptor mixture prepared above. The library wasprepared essentially according to the protocol outlined in the aboveLibrary Preparation User Guide, which is incorporated herein in itsentirety.

Template Preparation

A standard template protocol was performed as essentially described inthe Ion Template Kit User Guide (Life Technologies, Part No. 4469004),which is incorporated herein in its entirety.

Perform Standard Forward Sequencing

A standard sequencing protocol was performed as essentially described inthe Ion Sequencing 200 Kit User Guide (Life Technologies, Part No.4471998), which is incorporated herein in its entirety, except that thestandard sequencing primer was substituted for the paired-end sequencingprimer, prepared above. The paired-end sequencing primer was added tothe Ion Sphere Particles (ISPs) as follows:

Volume per chip Component Ion 314 ™ Chip Ion 316 ™ Chip Paired-endSequencing Primer 5 μL 12 μL Enriched ISPs in Annealing Buffer 8 μL 15μL Total volume 13 μL  27 μL

The standard sequencing protocol was then performed as essentiallyoutlined in the Sequencing Kit User Guide.

Denature the Sequencing Polymerase

After the sequencing run was complete, the Ion chip was removed from thePGM™ System and placed on a grounding plate or in an Ion centrifugeadapter/rotor bucket. While the Ion chip was removed, a dummy (used)chip was clamped into the PGM.

100 μL of EDS was applied into the loading port on the Ion chip. TheChip was then incubated at room temperature for 1 minute.

A 1× NEBuffer 2 was prepared by diluting the stock buffer 1:10 withnuclease-free water. A detergent, Tween-20 was also added to the bufferto a final concentration of 0.05%. The chip was then washed three timeswith 100 μL of 1× NEBuffer 2 containing Tween-20.

Fill in the Sequence

An extension solution was prepared as follows:

Volume per chip Ion 314 ™ Ion 316 ™ Component Chip Chip Nuclease-freewater 13 μL  52 μL  10 mM dNTPs (prepare a 1:4 dilution 2 μL 8 μL ofstock dNTPs from the Ion Xpress ™ Template 200 Kit) Tween 0.05% 1 μL 4μL 10X NEBuffer 2 2 μL 8 μL DNA Polymerase I, Large (Klenow) 2 μL 8 μLFragment Total 20 μL  80 μL 

The following volumes of extension solution were applied to the loadingport of the respective chip.

Ion 314™ Chip: 15 μl (˜5 μl overflow in flow cell wells)

Ion 316™ Chip: 75 μl (˜25 μl overflow in flow cell wells)

The chip was then transferred to a covered heating block (at 25° C.)containing a 50 ml tube cap filled with deionized water (to preventevaporation) and incubated for 10 minutes. After which, 100 ul of EDSwas loaded into the loading port, and incubated at room temperature for1 minute. After incubation, as much liquid as possible was removed fromthe loading port. A 1× NEBuffer 4 was prepared by diluting the stockbuffer 1:10 with nuclease-free water. A detergent, Tween-20, was alsoadded to the buffer to a final concentration of 0.05%. The chip was thenwashed three times with 100 μL of 1× NEBuffer 4 containing Tween-20.

Denature the Template

A second strand solution was prepared as outlined below.

Volume per chip Ion 314 ™ Ion 316 ™ Component Chip Chip Nuclease-freewater 42 μL  168 μL  NEBuffer 4 (1x) 6 μL 24 μL Nt.BbvCl 6 μL 24 μL T7Exonuclease 6 μL 24 μL Total 60 μL  240 μL 

The following amounts of the second strand solution was dispensed intothe respective Chip.

Ion 314™ Chip: 15 μl (˜5 μl overflow in flow cell wells)

Ion 316™ Chip: 75 μl (˜25 μl overflow in flow cell wells)

The chip was placed on a 1.5 ml freezer rack half filled with deionizedwater and incubated at room temperature for 20 minutes. The fluid in thechip was replaced from the loading port, and the second strand solutionwas re-applied to the chip for a second and third time, with therespective incubation times for a total incubation time of 60 minutes.After the final incubation, residual liquid was removed from the chipand 100 μl of EDS was applied to the loading port. The chip wasincubated at room temperature for 1 minute, after which the EDS solutionwas removed from the chip.

The chip was then washed 3 times with 100 μl of annealing buffer fromthe Ion Sequencing 200 Kit.

The sequencing polymerase from the Ion Sequencing 200 Kit was diluted inannealing buffer as shown below:

Volume per chip Component Ion 314 ™ Chip Ion 316 ™ Chip SequencingPolymerase 1.5 μL  6 μL Annealing Buffer   6 μL 24 μL Total volume 7.5μL 30 μL

Any residual annealing buffer from the wash steps was removed from thechip, and the diluted sequencing polymerase was applied (in full volume)to the loading port and incubated at room temperature for 5 minutes.During incubation, the sequencing key for the PGM system was altered asshown below:

U) From the main screen, press Options.

V) Press Advanced.

W) Press Change “Library Key Sequence”.

X) Enter the new key sequence: TCAGC.

Y) Press Back to return to the main screen.

Following incubation, the dummy (used) chip was removed from the PGMsystem and the prepared paired-end sequencing chip was loaded. Thesequencing experiment was initiated via pressing “experiment” on the PGMsystem. During the chip check, the “wetload” box was unchecked. The chipwashing and loading steps provided in the Ion Sequencing 200 Kit werealso obviated to proceed directly to selecting the experimentalconfiguration and performance of the sequencing run.

Example 13

In this non-limiting example, a paired-end library was prepared asfollows.

Prepare the Paired-End Adaptor Mixture

Paired-End Adaptor oligonucleotides were created to form a paired-endadapter mixture for use in the library preparation. Paired-end P1Adaptor oligonucleotide 3 and 4 contain an Nt.BbvCl nick site, whileAdaptor A oligonucleotides 3 and 4 complete the adaptors mixture. Alloligonucleotides were HPLC purified and subjected to sodium saltexchange. In the oligonucleotides * denotes a phosphorothioate bond. Inthe oligonucleotides “Y” denotes a C or a T nucleotide at that position.

Paired-end P1 5′-CCACTACGCCTCCGCTTT Adapter oligo 3CCTCTCTATGGGCAGTCGGTG ATCCTCAGC-3′ SEQ ID NO: 7 Paired-end P15′-GCTGAGGATCACCGACTG Adapter oligo 4 CCCATAGAGAGGAAAGCGGAGGCGTAGYGG*T*T-3′ SEQ ID NO: 8 Adapter A 5′-CCATCTCATCCCTGCGTG oligo 3TCTCCGACTCAG-3′ SEQ ID NO: 4 Adapter A 5′-CTGAGTCGGAGACACGCA oligo 4GGGATGAGATGG*T*T-3′ SEQ ID NO: 5

The following paired-end sequencing primer was ordered and purified asabove.

5′-C*C*A*T*CTCATCCCTGCGTGTCTCCGAC-3′ (SEQ ID NO: 6), wherein * denotes aphosphorothioate bond.

Prepare Enzyme Denaturation Solution

An enzyme denaturing solution (EDS) was prepared as follows for use inthe paired-end library: 10×TE pH 8.0 (5 ml), 20% SDS (5 ml) and 50 mMNaCl (0.5 ml) to a total of 50 ml in nuclease-free water. Finalconcentration: 1×TE pH 8.0, 2% SDS and 50 mM NaCl. 1.3 ml of EDS wasused per sequencing reaction.

Prepare Additive Solution

A 4% or 8% polyvinylpyrrolidone solution (PVP) was prepared as followsfor use in the paired-end library. Dissolve 0.4 grams of PVP40 into 4.8ml of nuclease-free water to a total volume of 5 ml (8% solution) ordissolve 0.2 grams of PVP40 into 4.8 ml of nuclease-free water to atotal volume of 5 ml (4% solution).

Prepare the Paired-End Adaptors Mixture

Each Adaptor oligonucleotide was diluted to a concentration of 100 umwith Y μl of nuclease-free water, determined as follows:X nmole oligo/100 nmole×1000=Y μl of nuclease-free water

The following reactions were prepared in separate sterile tubes:

Tube 1 Tube 2 Paired-end P1 Adapter oligo 3 (100 μM) 50 μL — Paired-endP1 Adapter oligo 4 (100 μM) 50 μL — A Adapter oligo 3 (100 μM) — 50 μL AAdapter oligo 4 (100 μM) — 50 μL T4 DNA Ligase Buffer (5X) 25 μL 25 μL

Each tube was heated using a thermal cycler as follows:

A) Heat at 90° C. for 2 minutes

B) Cool to room temperature (˜120 minutes)

Equal volumes of each adaptor were combined into a single tube to form acomplete adaptor mixture at a final concentration of about 20 μM. Thecomplete adaptor mixture was then stored at −20° C. until ready toprogress to the next step.

Library Preparation: Substitute the Paired-End Adaptors Mixture

A non-barcoded library was prepared as essentially described in the IonPlus Fragment Library Kit (Life Technologies, Part No. 4471252), exceptthe standard adaptors of the Ion Plus Fragment Library Kit weresubstituted with the adaptor mixture prepared above. The library wasprepared essentially according to the protocol outlined in the aboveLibrary Preparation User Guide, which is incorporated herein in itsentirety.

Template Preparation

A standard template protocol was performed as essentially described inthe Ion Template Kit User Guide (Life Technologies, Part No. 4469004),which is incorporated herein in its entirety.

Perform Standard Forward Sequencing

A standard sequencing protocol was performed as essentially described inthe Ion Sequencing 200 Kit User Guide (Life Technologies, Part No.4471998), which is incorporated herein in its entirety, except that thestandard sequencing primer was substituted for the paired-end sequencingprimer, prepared above. The paired-end sequencing primer was added tothe Ion Sphere Particles (ISPs) as follows:

Volume per chip Component Ion 314 ™ Chip Ion 316 ™ Chip Paired-endSequencing Primer 5 μL 12 μL Enriched ISPs in Annealing Buffer 8 μL 15μL Total volume 13 μL  27 μL

The standard sequencing protocol was then performed as essentiallyoutlined in the Sequencing Kit User Guide.

Denature the Sequencing Polymerase

After the sequencing run was complete, the Ion chip was removed from thePGM™ System and placed on a grounding plate or in an Ion centrifugeadapter/rotor bucket. While the Ion chip was removed, a dummy (used)chip was clamped into the PGM.

A 1× NEBuffer 2 was prepared by diluting the stock buffer 1:10 withnuclease-free water. An additive, PVP, was also added to the buffer to afinal concentration of 0.4%. The chip was then washed three times with100 μL of 1× NEBuffer 2 containing PVP.

Fill in the Sequence

An extension solution was prepared as follows:

Volume per chip Component Ion 314 ™ Chip Ion 316 ™ Chip Nuclease-freewater 13 μL  60 μL  10 mM dNTPs (prepare a 1:4 2 μL 8 μL dilution ofstock dNTPs from the Ion Xpress ™ Template 200 Kit) PVP 0.4% 1 μL 2 μL10X NEBuffer 2 2 μL 8 μL DNA Polymerase I, Large 2 μL 2 μL (Klenow)Fragment Total 20 μL  80 μL 

The following volumes of extension solution were applied to the loadingport of the respective chip.

Ion 314™ Chip: 15 μl (˜5 μl overflow in flow cell wells)

Ion 316™ Chip: 75 μl (˜25 μl overflow in flow cell wells)

The chip was then transferred to a covered heating block (at 25° C.)containing a 50 ml tube cap filled with deionized water (to preventevaporation) and incubated for 10 minutes. A 1× NEBuffer 4 was preparedby diluting the stock buffer 1:10 with nuclease-free water. An additive,PVP, was also added to the buffer to a final concentration of 0.4%. Thechip was then washed three times with 100 μL of 1× NEBuffer 4 containingPVP.

Denature the Template

A second strand solution was prepared as outlined below.

Volume per chip Component Ion 314 ™ Chip Ion 316 ™ Chip Nuclease-freewater 36 μL  144 μL  NEBuffer 4 (1x) 6 μL 24 μL Nt.BbvCl 6 μL 24 μL PVP0.4% 6 μL 24 μL T7 Exonuclease 6 μL 24 μL Total 60 μL  240 μL 

The following amounts of the second strand solution was dispensed intothe respective Chip.

Ion 314™ Chip: 15 μl (˜5 μl overflow in flow cell wells)

Ion 316™ Chip: 75 μl (˜25 μl overflow in flow cell wells)

The chip was placed on a 1.5 ml freezer rack half filled with deionizedwater and incubated at room temperature for 20 minutes. The fluid in thechip was replaced from the loading port, and the second strand solutionwas re-applied to the chip for a second and third time, with therespective incubation times for a total incubation time of 60 minutes.After the final incubation, residual liquid was removed from the chipand 100 μl of EDS was applied to the loading port. The chip wasincubated at room temperature for 1 minute, after which the EDS solutionwas removed from the chip.

The chip was then washed 3 times with 100 μl of annealing buffer fromthe Ion Sequencing 200 Kit.

The sequencing polymerase from the Ion Sequencing 200 Kit was diluted inannealing buffer as shown below:

Volume per chip Component Ion 314 ™ Chip Ion 316 ™ Chip SequencingPolymerase 1.5 μL  6 μL PVP 0.4%  1 μL  3 μL Annealing Buffer  5 μL 21μL Total volume 7.5 μL 30 μL

Any residual annealing buffer from the wash steps was removed from thechip, and the diluted sequencing polymerase was applied (in full volume)to the loading port and incubated at room temperature for 5 minutes.During incubation, the sequencing key for the PGM system was altered asshown below:

Z) From the main screen, press Options.

AA) Press Advanced.

BB) Press Change “Library Key Sequence”.

CC) Enter the new key sequence: TCAGC.

DD) Press Back to return to the main screen.

Following incubation, the dummy (used) chip was removed from the PGMsystem and the prepared paired-end sequencing chip was loaded. Thesequencing experiment was initiated via pressing “experiment” on the PGMsystem. During the chip check, the “wetload” box was unchecked. The chipwashing and loading steps provided in the Ion Sequencing 200 Kit werealso obviated to proceed directly to selecting the experimentalconfiguration and performance of the sequencing run.

Example 14

Sequencing data obtained on an Ion 314™ Chip (Life Technologies) usingthe paired-end methods of Example 8 and Example 9 are provided in FIGS.15A and 15B. Modifying Example 8 to reduce the number of EDS washesduring the paired-end sequencing protocol (i.e., Example 9) provided asignificant increase in the amount of paired sequencing runs (>75% ofreads are paired) and also 80% of the forward reads in the reversedirection were obtained at the same quality.

Example 15

The methodology of Example 9 was further modified to increase theconcentration of sequencing polymerase in the paired-end sequencingprotocol. S 16 provides data from such analysis using Ion 314™ Chips(Life Technologies). Increasing the concentration of sequencingpolymerase (2-fold) was found to result in an increase in paired readsand increase in the quality of reverse reads.

Example 16

The methodology of Example 10 was further modified to increase theconcentration of sequencing polymerase in the paired-end sequencingprotocol. FIGS. 17A and 17B provide data from such analysis using Ion314™ Chips (Life Technologies). Increasing the concentration ofsequencing polymerase (2-fold) and removing the single EDS wash wasfound to result in an increase in paired reads and increase in thequality of reverse reads.

Example 17

FIGS. 18A, 18B and 18C provide data obtained from Examples 11 and 12.Example 11 included the presence of an additive in the buffer, whileExample 12 included the presence of a detergent in the buffer. The dataprovided in FIGS. 18A, 18B and 18C were obtained using Ion 314™ Chips(Life Technologies). The introduction of a detergent, such as 0.05%Tween-20, into the sequencing buffer increased paired-reads and qualityof reverse reads. Addition of an additive, such as 0.4% PVP, into thesequencing buffer was also found to increase the number of paired readsand quality of reverse reads. PVP or Tween-20 in the sequencing bufferwas found to result in >75% loading density of the Ion 314™ Chips.

Example 18

FIGS. 19A, 19B and 19C provide data obtained using the protocols ofExample 9, Example 10, or Example 11, using Ion 314™ Chips (LifeTechnologies). Removing the single EDS wash step from the paired endsequencing protocol was found to result in an increase in paired readsand increase in the quality of reverse reads (Example 9 as compared toExample 10). Additionally, the introduction of an additive, such as 0.4%PVP, was also found to increase the number of paired reads and qualityof reverse reads (Example 9 or Example 10 as compared to Example 11).

Example 19

In this example, the sequencing run was a 2×200 base pair paired-end runusing a 300 bp insert from a Rhodopseudomonas palustris CGA009 Library.FIGS. 20A and 20B provide data from such analysis using Ion 316™ Chips(Life Technologies). FIGS. 20A and 20B provide data obtained using theprotocols of Example 9 or Example 11. Reducing or removing the EDS washsteps from the paired end sequencing protocol was found to result in anincrease in paired reads and increase in the quality of reverse reads.Additionally, the introduction of an additive, such as 0.4% PVP was alsofound to increase the number of paired reads and quality of reversereads.

Example 20

In this example, the sequencing run was a 2×200 base pair paired-end runusing a 300 bp insert from a Rhodopseudomonas palustris CGA009 Library.FIG. 21 provides data from such analysis using Ion 316™ Chips (LifeTechnologies). FIG. 21A provides data obtained using the protocol ofExample 13. The protocol of Example 13 was also amended to modify theincubation time of the sequencing enzyme from 5 minutes to 30 minutes(FIG. 21B). Increasing the incubation time of the sequencing enzyme from5 to 30 minutes was found to result in an increase in paired reads andincrease in the quality of reverse reads.

Example 21

In this non-limiting example, a paired-end library was prepared asfollows.

Prepare the Paired-End Adaptor Mixture

Paired-End Adaptor oligonucleotides were created to form a paired-endadapter mixture for use in the library preparation. Paired-end P1Adaptor oligonucleotide 3 and 4 contain an Nt.BbvCl nick site, whileAdaptor A oligonucleotides 3 and 4 complete the adaptors mixture. Alloligonucleotides were HPLC purified and subjected to sodium saltexchange. In the oligonucleotides * denotes a phosphorothioate bond. Inthe oligonucleotides “Y” denotes a C or a T nucleotide at that position.

Paired-end P1 5′-CCACTACGCCTCCGCTTT Adapter oligo 3CCTCTCTATGGGCAGTCGGTG ATCCTCAGC-3′ SEQ ID NO: 7 Paired-end P15′-GCTGAGGATCACCGACTG Adapter oligo 4 CCCATAGAGAGGAAAGCGGAGGCGTAGYGG*T*T-3′ SEQ ID NO: 8 Adapter A 5′-CCATCTCATCCCTGCGTG oligo 3TCTCCGACTCAG-3′ SEQ ID NO: 4 Adapter A 5′-CTGAGTCGGAGACACGCA oligo 4GGGATGAGATGG*T*T-3′ SEQ ID NO: 5

The following paired-end sequencing primer was ordered and purified asabove.

5′-C*C*A*T*CTCATCCCTGCGTGTCTCCGAC-3′ (SEQ ID NO: 6), wherein * denotes aphosphorothioate bond.

Prepare Enzyme Denaturation Solution

An enzyme denaturing solution (EDS) was prepared as follows for use inthe paired-end library: 10×TE pH 8.0 (5 ml), 20% SDS (5 ml) and 50 mMNaCl (0.5 ml) to a total of 50 ml in nuclease-free water. Finalconcentration: 1×TE pH 8.0, 2% SDS and 50 mM NaCl. 1.3 ml of EDS wasused per sequencing reaction.

Prepare Additive Solution

A 8% polyvinylpyrrolidone solution (PVP) was prepared as follows for usein the paired-end library. Dissolve 0.4 grams of PVP40 into 4.8 ml ofnuclease-free water to a total volume of 5 ml (8% solution).

Prepare the Paired-End Adaptors Mixture

Each Adaptor oligonucleotide was diluted to a concentration of 100 umwith Y μl of nuclease-free water, determined as follows:X nmole oligo/100 nmole×1000=Y μl of nuclease-free water

The following reactions were prepared in separate sterile tubes:

Tube 1 Tube 2 Paired-end P1 Adapter oligo 3 (100 μM) 50 μL — Paired-endP1 Adapter oligo 4 (100 μM) 50 μL — A Adapter oligo 3 (100 μM) — 50 μL AAdapter oligo 4 (100 μM) — 50 μL T4 DNA Ligase Buffer (5X) 25 μL 25 μL

Each tube was heated using a thermal cycler as follows:

A) Heat at 90° C. for 2 minutes

B) Cool to room temperature (˜120 minutes)

Equal volumes of each adaptor were combined into a single tube to form acomplete adaptor mixture at a final concentration of about 20 μM. Thecomplete adaptor mixture was then stored at −20° C. until ready toprogress to the next step.

Library Preparation: Substitute the Paired-End Adaptors Mixture

A non-barcoded library was prepared as essentially described in the IonPlus Fragment Library Kit (Life Technologies, Part No. 4471252), exceptthe standard adaptors of the Ion Plus Fragment Library Kit weresubstituted with the adaptor mixture prepared above. The library wasprepared essentially according to the protocol outlined in the aboveLibrary Preparation User Guide, which is incorporated herein in itsentirety.

Template Preparation

A standard template protocol was performed as essentially described inthe Ion Template Kit User Guide (Life Technologies, Part No. 4469004),which is incorporated herein in its entirety.

Perform Standard Forward Sequencing

A standard sequencing protocol was performed as essentially described inthe Ion Sequencing 200 Kit User Guide (Life Technologies, Part No.4471998), which is incorporated herein in its entirety, except that thestandard sequencing primer was substituted for the paired-end sequencingprimer, prepared above. The paired-end sequencing primer was added tothe Ion Sphere Particles (ISPs) as follows:

Volume per chip Component Ion 314 ™ Chip Ion 316 ™ Chip Paired-endSequencing Primer 5 μL 12 μL Enriched ISPs in Annealing Buffer 8 μL 15μL Total volume 13 μL  27 μL

The standard sequencing protocol was then performed as essentiallyoutlined in the Sequencing Kit User Guide.

Denature the Sequencing Polymerase

After the sequencing run was complete, the Ion chip was removed from thePGM™ System and placed on a grounding plate or in an Ion centrifugeadapter/rotor bucket. While the Ion chip was removed, a dummy (used)chip was clamped into the PGM.

A 1× NEBuffer 2 was prepared by diluting the stock buffer 1:10 withnuclease-free water. An additive, PVP, was also added to the buffer to afinal concentration of 0.4%. The chip was then washed twice with 100 μLof 1× NEBuffer 2 containing PVP.

Fill in the Sequence

An extension solution was prepared as follows:

Volume per chip Component Ion 314 ™ Chip Ion 316 ™ Chip Nuclease-freewater 13 μL  52 μL  10 mM dNTPs (prepare a 1:4 2 μL 8 μL dilution ofstock dNTPs from the Ion Xpress ™ Template 200 Kit) PVP 8% 1 μL 4 μL 10XNEBuffer 2 2 μL 8 μL DNA Polymerase I, Large 2 μL 8 μL (Klenow) FragmentTotal 20 μL  80 μL 

The following volumes of extension solution were applied to the loadingport of the respective chip.

Ion 314™ Chip: 15 μl (˜5 μl overflow in flow cell wells)

Ion 316™ Chip: 75 μl (˜25 μl overflow in flow cell wells)

The chip was then transferred to a covered heating block (at 25° C.)containing a 50 ml tube cap filled with deionized water (to preventevaporation) and incubated for 10 minutes. 100 μl of EDS was applied tothe loading port. A 1× NEBuffer 4 was prepared by diluting the stockbuffer 1:10 with nuclease-free water. An additive, PVP, was also addedto the buffer to a final concentration of 0.4%. The chip was then washedtwice with 100 μL of 1× NEBuffer 4 containing PVP.

Denature the Template

A second strand solution was prepared as outlined below.

Volume per chip Component Ion 314 ™ Chip Ion 316 ™ Chip Nuclease-freewater 39 μL  156 μL  NEBuffer 4 (10x) 6 μL 24 μL Nt.BbvCl 6 μL 24 μL PVP8% 3 μL 12 μL T7 Exonuclease 6 μL 24 μL Total 60 μL  240 μL 

The following amounts of the second strand solution was dispensed intothe respective Chip.

Ion 314™ Chip: 15 μl (˜5 μl overflow in flow cell wells)

Ion 316™ Chip: 75 μl (˜25 μl overflow in flow cell wells)

The chip was placed on a 1.5 ml freezer rack half filled with deionizedwater and incubated at room temperature for 20 minutes. Afterincubation, the fluid in the chip was replaced from the loading port,and the second strand solution was re-applied to the chip for a secondand third time, with the respective incubation times for a totalincubation time of 60 minutes. After the final incubation, residualliquid was removed from the chip and 100 μl of EDS was applied to theloading port. The chip was incubated at room temperature for 1 minute,after which the EDS solution was removed from the chip.

The chip was then washed twice with 100 μl of annealing buffer from theIon Sequencing 200 Kit, that was supplemented with PVP as follows:annealing buffer from the Ion Sequencing 200 kit (240 μl) was added to8% PVP40 (12 μl).

Perform Reverse Sequencing

The sequencing polymerase from the Ion Sequencing 200 Kit was diluted inannealing buffer as shown below:

Volume per chip Component Ion 314 ™ Chip Ion 316 ™ Chip SequencingPolymerase 1.5 μL  6 μL Annealing Buffer  6 μL 24 μL Total volume 7.5 μL30 μL

Any residual annealing buffer from the wash steps was removed from thechip, and the diluted sequencing polymerase was applied to the loadingport and incubated at room temperature for 5 minutes. For a 314™ Chip, 6μL of diluted polymerase was added. For a 316™ Chip, 25 μL of dilutedpolymerase was added. During incubation, the sequencing key for the PGMsystem was altered as shown below:

EE) From the main screen, press Options.

FF) Press Advanced.

GG) Press Change “Library Key Sequence”.

HH) Enter the new key sequence: TCAGC.

II) Press Back to return to the main screen.

Following incubation, the dummy (used) chip was removed from the PGMsystem and the prepared paired-end sequencing chip was loaded. Thesequencing experiment was initiated via pressing “experiment” on the PGMsystem. During the chip check, the “wetload” box was unchecked. The chipwashing and loading steps provided in the Ion Sequencing 200 Kit werealso obviated to proceed directly to selecting the experimentalconfiguration and performance of the sequencing run. The sequencing runwas performed as described in the Ion Sequencing 200 Kit User Guide

Example 22

In this exemplary embodiment, 11 independent paired-end sequencingreactions were performed as outlined in Example 21 with the exception ofusing 318 Ion Chips (Part No. Sequencing was performed using a PGMsequencer. The forward and reverse sequencing data obtained is providedin the table below. In each independent experiment, the forward read wasfound to produce over 1 gigabyte (Gb) of sequencing data at AQ20. Insome instances, the sum of the forward and reverse read were found toproduce over 2 Gb of sequencing data at AQ20

318 AQ20 throughput (Mb) Runs #1 #2 #3 #4 #5 #6 #7 forward 1,388.821,247.06 997.64 1,237.21 1,289.28 1,445.38 1,341.66 Reverse 723.44705.02 774.2 570.23 614.14 627.88 950.27 sum 2112.26 1952.08 1771.841807.44 1903.42 2073.26 2291.93 AQ17 AQ20 Read throughput length 318AQ20 throughput (Mb) Mean Mean Runs #8 #9 #10 #11 (MB) (bp) forward1204.55 1204.55 1204.55 1289.95 1259.15 216 Reverse 901.19 618.75 669.33783.67 721.65 166 sum 2105.74 1823.3 1873.88 2073.62 1,980.80

Example 23

In this non-limiting example, a paired-end library was prepared asfollows:

Prepare the Paired-End Adaptor Mixture

Paired-End Adaptor oligonucleotides were created to form a paired-endadapter mixture for use in the library preparation. Paired-end P1Adaptor oligonucleotide 3 and 4 contain an Nt.BbvCl nick site, whileAdaptor A oligonucleotides 3 and 4 complete the adaptors mixture. Alloligonucleotides were HPLC purified and subjected to sodium saltexchange. In the oligonucleotides * denotes a phosphorothioate bond.

Paired-end P1 5′-CCACTACGCCTCCGCTTT Adapter oligo 3CCTCTCTATGGGCAGTCGGTG ATCCTCAGC-3′ SEQ ID NO: 7 Paired-end P15′-GCTGAGGATCACCGACTG Adapter oligo 4 CCCATAGAGAGGAAAGCGGAGGCGTAGTGG*T*T-3′ SEQ ID NO: 9 Adapter A 5′-CCATCTCATCCCTGCGTG oligo 3TCTCCGACTCAG-3′ SEQ ID NO: 4 Adapter A 5′-CTGAGTCGGAGACACGCA oligo 4GGGATGAGATGG*T*T-3′ SEQ ID NO: 5

The following paired-end sequencing primer was ordered and purified asabove.

5′-C*C*A*T*CTCATCCCTGCGTGTCTCCGAC-3′ (SEQ ID NO: 6), wherein * denotes aphosphorothioate bond.

Prepare the Paired-End Adaptors Mixture

Each Adaptor oligonucleotide was diluted to a concentration of 100 umwith Y μl of nuclease-free water, determined as follows:X nmole oligo/100 nmole×1000=Y μl of nuclease-free water

The following reactions were prepared in separate sterile tubes:

Tube 1 Tube 2 Paired-end P1 Adapter oligo 3 (100 μM) 50 μL — Paired-endP1 Adapter oligo 4 (100 μM) 50 μL — A Adapter oligo 3 (100 μM) — 50 μL AAdapter oligo 4 (100 μM) — 50 μL T4 DNA Ligase Buffer (5X) 25 μL 25 μL

Each tube was heated using a thermal cycler as follows:

A) Heat at 90° C. for 2 minutes

B) Cool to room temperature (˜120 minutes)

Equal volumes of each adaptor were combined into a single tube to form acomplete adaptor mixture at a final concentration of about 20 μM. Thecomplete adaptor mixture was then stored at −20° C. until ready toprogress to the next step.

Library Preparation: Substitute the Paired-End Adaptors Mixture

A non-barcoded library was prepared as essentially described in the IonPlus Fragment Library Kit (Life Technologies, Part No. 4471252), exceptthe standard adaptors of the Ion Plus Fragment Library Kit weresubstituted with the adaptor mixture prepared above. The library wasprepared essentially according to the protocol outlined in the aboveLibrary Preparation User Guide, which is incorporated herein in itsentirety.

Template Preparation

A standard template protocol was performed as essentially described inthe Ion Express™ Template 200 Kit (Life Technologies, Part No. 4471253),which is incorporated herein in its entirety. For automated preparation,use the Ion One Touch™ Template Kit (Part No. 4468660).

Perform Standard Forward Sequencing

A standard sequencing protocol was performed as essentially described inthe Ion Sequencing 200 Kit User Guide (Life Technologies, Part No.4471998), which is incorporated herein in its entirety, except that thestandard sequencing primer was substituted for the paired-end sequencingprimer, prepared above. The paired-end sequencing primer was added tothe Ion Sphere Particles (ISPs) as follows:

Volume per chip Component Ion 314 ™ Chip Ion 316 ™ Chip Paired-endSequencing Primer 5 μL 12 μL Enriched ISPs in Annealing Buffer 8 μL 15μL Total volume 13 μL  27 μL

The standard sequencing protocol was then performed as essentiallyoutlined in the Sequencing Kit User Guide.

Denature the Sequencing Polymerase

After the sequencing run was complete, the Ion chip was removed from thePGM™ System and placed on a grounding plate or in an Ion centrifugeadapter/rotor bucket. While the Ion chip was removed, a dummy (used)chip was clamped into the PGM.

A 1× NEBuffer 2 (Fill-in Buffer) was prepared by diluting the stockbuffer 1:10 with nuclease-free water. The chip was then washed twicewith 100 μL of 1× NEBuffer 2.

Fill in the Sequence

An extension solution was prepared as follows:

Component Ion 318 ™ Chip Nuclease-free water 51 μL 10 mM dNTPs (preparea 1:4 dilution  8 μL of stock dNTPs from the Ion Xpress ™ Template 200Kit) 5X Fill-in Buffer 16 μL DNA Polymerase  5 μL Total 80 μL

The following volumes of extension solution were applied to the loadingport of the Ion 318™ Chip: 75 μl.

The chip was then transferred to a covered heating block (at 22° C.)containing a 50 ml tube cap filled with deionized water (to preventevaporation) and incubated for 10 minutes. 100 μl of EDS was applied tothe loading port. A 1× NEBuffer 4 was prepared by diluting the stockbuffer 1:10 with nuclease-free water. An additive, PVP, was also addedto the buffer to a final concentration of 0.4%. The chip was then washedtwice with 100 μL of 1× NEBuffer 4 containing PVP.

Denature the Template

A second strand solution was prepared as outlined below.

Component Ion 318 ™ Chip Nuclease-free water 104 μL Second strand buffer5x  32 μL Nickase  24 μL Total 160 μL

The following amount of the second strand solution was dispensed intothe Ion 318™ Chip: 75 μl.

The chip was placed into a thermocycler at 37° C. and incubated for 15minutes. After incubation, the fluid in the chip was replaced from theloading port, and the second strand solution was re-applied to the chipfor a second time, with the respective incubation time for a total of 30minutes.

During the second incubation, either a 100 mM DTT solution or a 4 mMTCEP solution was prepared and adding to a 1× NEBuffer 4.

After incubation, the residual liquid was removed from the chip and 100μl of EDS was applied to the loading port. The chip was incubated atroom temperature for 1 minute, after which the EDS solution was removedfrom the chip. The chip was washed three times with the 1× NEBuffer 4containing fresh DTT or TCEP. The chip was then incubated at 22° C. for30 minutes. After incubation, the residual liquid was removed from thechip and 100 μl of EDS was applied to the loading port. The chip wasincubated at room temperature for 1 minute.

Perform Reverse Sequencing

The sequencing polymerase from the Ion Sequencing 200 Kit was diluted inannealing buffer as shown below:

Component Ion 316 ™ Chip Sequencing Polymerase  6 μL Annealing Buffer 26μL Total volume 32 μL

After completing the 1 min incubation, the chip was washed three timeswith annealing buffer. Any residual annealing buffer from the wash stepswas removed from the chip, and the diluted sequencing polymerase wasapplied to the loading port and incubated at room temperature for 5minutes.

Following incubation, the dummy (used) chip was removed from the PGMsystem and the prepared paired-end sequencing chip was loaded. Thesequencing experiment was initiated via pressing “experiment” on the PGMsystem.

Example 24

Comparative data showing the effects of reducing agents DTT or TCEP onT7 exonuclease digestion using the protocol provided in Example 23 isprovided in FIG. 22. Sequencing data obtained on a 318™ Ion Chip using 4mM TCEP and the protocol of Example 23 provided a reverse/forward (%) ofgreater than 93%.

Example 25

Additional methodologies were evaluated as a means to reduce digestionof the bead-immobilized primer (Primer B) during exonuclease treatmentin exemplary embodiments of the paired end sequencing methods accordingto the disclosure. It was determined that the following methods can beused to reduce bead-immobilized primer digestion or solid-supportimmobilized primer digestion during paired end sequencing. As detailedin Example 26, the inclusion of additional phosphorothioate residuesinto the immobilized primer (primer B) were found to reduce the level ofimmobilized primer digestion during paired end sequencing. Additionally,the presence of additional phosphorothioate residues in the immobilizedprimer as compared to the standard immobilized (four phosphorothioate)primer was found to increase sequencing throughput (AQ20), percentagepairing, and percentage reverse/forward reads (FIGS. 23A, 23B, 23C, 23D,23E and 23F). Thus, the method can be used to improve nucleic acidsequencing throughput and percentage of paired end sequencing reads.

A C18 polyethylene glycol (PEG) linker was also evaluated as a substratefor reducing digestion of the immobilized primer during paired endsequencing. A blunt-ended substrate (Oligo 753) was created and comparedto the equivalent substrate with an appended C18 PEG linker (Oligo 777).FIG. 24 shows data obtained during exonuclease incubation of the twooligonucleotide substrates. As can be seen, the presence of a PEG linkerdid not significantly differ from the data obtained with the blunt endedsubstrate; however substantial inhibition of exonuclease activity wasobtained with the subsequent modifications.

A poly-T stretch of nucleotides appended to Oligo 753 was also evaluatedas a substrate for reducing exonuclease digestion of immobilized primersduring paired end sequencing. The blunt-ended substrate (Oligo 753) wascompared to an equivalent substrate having a poly-T stretch of 5 Tnucleotides (Oligo 779). FIG. 25 shows data obtained during exonucleaseincubation of the two oligonucleotide substrates. As can be seen, thepoly-T stretch of 5 nucleotides reduced exonuclease digestion by about45%.

A poly-T stretch of 10 or 15 nucleotides appended to Oligo 753 were alsoevaluated as substrates for reducing exonuclease digestion ofimmobilized primers during paired end sequencing. The blunt-endedsubstrate (Oligo 753) was compared to an equivalent substrate appendedwith a poly-T stretch of 10 T nucleotides (Oligo 783) or a poly-Tstretch of 15 nucleotides (Oligo 784). FIG. 26 shows data obtainedduring exonuclease incubation of the 10 and 15 poly-T substrates ascompared to the substrate containing a poly-T stretch of 5 nucleotides(Oligo 779). As can be seen, increasing the length of the poly-T stretchof nucleotides substantially reduces the rate of exonuclease digestion.Overall, it was observed that an oligonucleotide substrate with astretch of 10 T nucleotides was hydrolyzed at a rate of about 10%compared to the equivalent blunt-ended substrate.

Example 26

In this example, a comparative paired end sequencing study wasundertaken using either the Standard Operating Procedure (SOP) primerthat contains four phosphorothioate residues (e.g., SEQ ID NO: 6) ormodifying the sequencing primer to include five or six phosphorothioateresidues (e.g., SEQ ID NO: 2). The paired end sequencing data obtainedwith five phosphorothioate residues according to the protocol of Example23 is presented in FIGS. 23A, 23B, 23C, 23D, 23E and 23F.

Example 27

In this example, a modification to block polymerase extension (e.g.,during the fill-in reaction of the paired end sequencing methods) wasevaluated. A standard polymerase oligonucleotide substrate was preparedas a control (Oligo 221) and compared against equivalent substrates thateither contained: a) a stretch of 5 T nucleotides (Oligo 779); b) astretch of 15 T nucleotides (Oligo 784); or c) a stretch of 15 Tnucleotides preceded by an abasic site (Oligo 786). FIG. 27 shows dataobtained during polymerase extension reactions for each oligonucleotidesubstrate. An abasic site in conjunction with a stretch of 15 Tnucleotides was found to significantly block polymerase extension. Anexonuclease digestion test was also performed according to Example 25 toevaluate the abasic site/poly-T substrate. It was determined that theabasic site/poly-T substrate did not increase the rate of exonucleasedigestion compared to the poly-T 15 nucleotide substrate.

While the principles of the present teachings have been described inconnection with specific embodiments, it should be understood clearlythat these descriptions are made only by way of example and are notintended to limit the scope of the present teachings or claims. What hasbeen disclosed herein has been provided for the purposes of illustrationand description. It is not intended to be exhaustive or to limit what isdisclosed to the precise forms described. Many modifications andvariations will be apparent to the practitioner skilled in the art. Whatis disclosed was chosen and described in order to best explain theprinciples and practical application of the disclosed embodiments of theart described, thereby enabling others skilled in the art to understandthe various embodiments and various modifications that are suited to theparticular use contemplated. It is intended that the scope of what isdisclosed be defined by the following claims and their equivalents.

What is claimed:
 1. A method for generating single-stranded nucleicacids, comprising: a) hybridizing a first primer to a nucleic acidmolecule; b) extending the first primer via polymerization underpolymerization conditions, thereby forming an extended first primerproduct that is complementary to a portion of the nucleic acid molecule;c) introducing a nick into a portion of the nucleic acid molecule thatis hybridized to the extended first primer product, where the nickincludes a free 5′ end and a free 3′ end in the nucleic acid molecule;and d) degrading the nucleic acid molecule from the free 5′ end of thenick using a degrading agent, thereby generating a single-strandedextended primer product which remains hybridized to an undegradedportion of the nucleic acid molecule.
 2. The method of claim 1, whereinthe nucleic acid molecule is a single stranded nucleic acid template. 3.The method of claim 1, wherein the 5′ end of the nucleic acid moleculeis linked to a solid support.
 4. The method of claim 1, wherein thesolid support comprises a bead, particle, microparticle, microsphere,slide, flowcell or reaction chamber.
 5. The method of claim 1, whereinthe nucleic acid molecule includes a scissile linkage.
 6. The method ofclaim 1, wherein the nick is introduced into a portion of the nucleicacid molecule with a nicking enzyme or restriction enzyme.
 7. The methodof claim 1, wherein the nick is introduced by nicking a scissile linkagewithin the nucleic acid molecule, wherein the scissile linkage comprisesa phosphorothioate linkage, phosphoramidate linkage, photolabileinternucleosidic linkage, apurinic tetrahydrofuran site, or uracil base.8. The method of claim 1, further comprising: a) separating thesingle-stranded extended primer product from the undegraded portion ofthe nucleic acid molecule; b) capturing the separated single-strandedextended primer product onto a solid support; and c) sequencing theseparated single-stranded extended primer product.
 9. The method ofclaim 8, wherein the solid support comprises a bead, microsphere,nanopore, well, trough, groove, channel, flowcell, microwell, slide orother attachment surface.